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Sample records for nonmuscle myosin ii-b

  1. P-cadherin counteracts myosin II-B function: implications in melanoma progression

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    De Wever Olivier

    2010-09-01

    Full Text Available Abstract Background Malignant transformation of melanocytes is frequently attended by a switch in cadherin expression profile as shown for E- and N-cadherin. For P-cadherin, downregulation in metastasizing melanoma has been demonstrated, and over-expression of P-cadherin in melanoma cell lines has been shown to inhibit invasion. The strong invasive and metastatic nature of cutaneous melanoma implies a deregulated interplay between intercellular adhesion and migration-related molecules Results In this study we performed a microarray analysis to compare the mRNA expression profile of an invasive BLM melanoma cell line (BLM LIE and the non-invasive P-cadherin over-expression variant (BLM P-cad. Results indicate that nonmuscle myosin II-B is downregulated in BLM P-cad. Moreover, myosin II-B plays a major role in melanoma migration and invasiveness by retracting the tail during the migratory cycle, as shown by the localization of myosin II-B stress fibers relative to Golgi and the higher levels of phosphorylated myosin light chain. Analysis of P-cadherin and myosin II-B in nodular melanoma sections and in a panel of melanoma cell lines further confirmed that there is an inverse relationship between both molecules. Conclusions Therefore, we conclude that P-cadherin counteracts the expression and function of myosin II-B, resulting in the suppression of the invasive and migratory behaviour of BLM melanoma cells

  2. Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster.

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    Heissler, Sarah M; Chinthalapudi, Krishna; Sellers, James R

    2015-04-01

    Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.

  3. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

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    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  4. Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

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    Qiu Xinping

    2010-03-01

    Full Text Available Abstract Background Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear. Results In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II. Conclusion Our results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

  5. Nonmuscle myosin II isoforms coassemble in living cells.

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    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

  6. Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

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    Billington, Neil; Beach, Jordan R; Heissler, Sarah M; Remmert, Kirsten; Guzik-Lendrum, Stephanie; Nagy, Attila; Takagi, Yasuharu; Shao, Lin; Li, Dong; Yang, Yi; Zhang, Yingfan; Barzik, Melanie; Betzig, Eric; Hammer, John A; Sellers, James R

    2015-03-30

    Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

  7. Life without double-headed non-muscle myosin II motor proteins

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    Venkaiah eBetapudi

    2014-07-01

    Full Text Available Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  8. Life without double-headed non-muscle myosin II motor proteins

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    Betapudi, Venkaiah

    2014-07-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  9. Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

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    Qiu Xinping; Seabrooke Sara; Stewart Bryan A

    2010-01-01

    Abstract Background Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear. Results In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic ves...

  10. Memory Disrupting Effects of Nonmuscle Myosin II Inhibition Depend on the Class of Abused Drug and Brain Region

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    Briggs, Sherri B.; Blouin, Ashley M.; Young, Erica J.; Rumbaugh, Gavin; Miller, Courtney A.

    2017-01-01

    Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared…

  11. Memory Disrupting Effects of Nonmuscle Myosin II Inhibition Depend on the Class of Abused Drug and Brain Region

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    Briggs, Sherri B.; Blouin, Ashley M.; Young, Erica J.; Rumbaugh, Gavin; Miller, Courtney A.

    2017-01-01

    Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared…

  12. Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease

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    Pecci, A.; Panza, E.; Pujol-Moix, N.

    2008-01-01

    MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness...

  13. Role of LARP6 and nonmuscle myosin in partitioning of collagen mRNAs to the ER membrane.

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    Hao Wang

    Full Text Available Type I collagen is extracellular matrix protein composed of two α1(I and one α2(I polypeptides that fold into triple helix. Collagen polypeptides are translated in coordination to synchronize the rate of triple helix folding to the rate of posttranslational modifications of individual polypeptides. This is especially important in conditions of high collagen production, like fibrosis. It has been assumed that collagen mRNAs are targeted to the membrane of the endoplasmic reticulum (ER after translation of the signal peptide and by signal peptide recognition particle (SRP. Here we show that collagen mRNAs associate with the ER membrane even when translation is inhibited. Knock down of LARP6, an RNA binding protein which binds 5' stem-loop of collagen mRNAs, releases a small amount of collagen mRNAs from the membrane. Depolimerization of nonmuscle myosin filaments has a similar, but stronger effect. In the absence of LARP6 or nonmuscle myosin filaments collagen polypeptides become hypermodified, are poorly secreted and accumulate in the cytosol. This indicates lack of coordination of their synthesis and retro-translocation due to hypermodifications and misfolding. Depolimerization of nonmuscle myosin does not alter the secretory pathway through ER and Golgi, suggesting that the role of nonmuscle myosin is primarily to partition collagen mRNAs to the ER membrane. We postulate that collagen mRNAs directly partition to the ER membrane prior to synthesis of the signal peptide and that LARP6 and nonmuscle myosin filaments mediate this process. This allows coordinated initiation of translation on the membrane bound collagen α1(I and α2(I mRNAs, a necessary step for proper synthesis of type I collagen.

  14. Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

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    Kriajevska, M; Bronstein, I B; Scott, D J

    2000-01-01

    A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays...

  15. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

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    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  16. Non-muscle myosin as target antigen for human autoantibodies in patients with hepatitis C virus-associated chronic liver diseases.

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    von Mühlen, C A; Chan, E K; Peebles, C L; Imai, H; Kiyosawa, K; Tan, E M

    1995-04-01

    Three patients with hepatitis C virus (HCV)-related chronic liver disease were shown to have autoantibodies strongly reacting with cytoskeletal fibres of non-muscle cells. The heavy chain of non-muscle myosin microfilament was the main target for those autoantibodies, as determined by (i) cell and tissue immunofluorescence studies showing colocalization with an anti-myosin antibody prototype; (ii) primary reactivity in immunoblotting with a 200-kD protein, using either MOLT-4 cells, human platelets, or affinity-purified non-muscle myosin as antigen extract; and (iii) immunoblotting of similar immunoreactive fragments in papain-digested MOLT-4 cell extracts, by using those human sera and antibody prototype. Autoantibodies to non-muscle myosin heavy chain were not previously reported in patients with chronic liver diseases, especially in those associated with HCV infection.

  17. SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

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    Fan, Xueping; Yang, Hongying; Kumar, Sudhir; Tumelty, Kathleen E.; Pisarek-Horowitz, Anna; Sharma, Richa; Chan, Stefanie; Tyminski, Edyta; Shamashkin, Michael; Belghasem, Mostafa; Henderson, Joel M.; Coyle, Anthony J.; Berasi, Stephen P.

    2016-01-01

    The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss. PMID:27882344

  18. Non-muscle Myosin II Isoforms Co-assemble in Living Cells

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    Beach, Jordan R.; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A.

    2014-01-01

    SUMMARY Non-muscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB and IIC), each of which possesses distinct biophysical properties and supports unique, as well as redundant, cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear if NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or if filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged versions of NM IIA, IIB and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms co-assemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly-spread cells, arguing for the existence of sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NM II isoforms. PMID:24814144

  19. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

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    Morin, Nicole A; Oakes, Patrick W; Hyun, Young-Min; Lee, Dooyoung; Chin, Y Eugene; Chin, Eugene Y; King, Michael R; Springer, Timothy A; Shimaoka, Motomu; Tang, Jay X; Reichner, Jonathan S; Kim, Minsoo

    2008-01-21

    Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

  20. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities

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    Karen A. Newell-Litwa

    2015-12-01

    Full Text Available The actin motor protein non-muscle myosin II (NMII acts as a master regulator of cell morphology, with a role in several essential cellular processes, including cell migration and post-synaptic dendritic spine plasticity in neurons. NMII also generates forces that alter biochemical signaling, by driving changes in interactions between actin-associated proteins that can ultimately regulate gene transcription. In addition to its roles in normal cellular physiology, NMII has recently emerged as a critical regulator of diverse, genetically complex diseases, including neuronal disorders, cancers and vascular disease. In the context of these disorders, NMII regulatory pathways can be directly mutated or indirectly altered by disease-causing mutations. NMII regulatory pathway genes are also increasingly found in disease-associated copy-number variants, particularly in neuronal disorders such as autism and schizophrenia. Furthermore, manipulation of NMII-mediated contractility regulates stem cell pluripotency and differentiation, thus highlighting the key role of NMII-based pharmaceuticals in the clinical success of stem cell therapies. In this Review, we discuss the emerging role of NMII activity and its regulation by kinases and microRNAs in the pathogenesis and prognosis of a diverse range of diseases, including neuronal disorders, cancer and vascular disease. We also address promising clinical applications and limitations of NMII-based inhibitors in the treatment of these diseases and the development of stem-cell-based therapies.

  1. Species Differences in the Distribution of the Nonmuscle Myosin Heavy Chain IIB Inserted Isoform in the Brain(Biochemistry)

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    Shin-ya, Hagiwara; MASAYUKI, TAKAHASHI; Akihiko, Yamagishi; Division of Biological Sciences, Graduate School of Science, Hokkaido University

    2001-01-01

    The alternatively spliced isoform of the nonmuscle myosin heavy chain IIB (MHC-IIB) with an insert of 21 amino acids near the actin-binding region, MHC-IIB (B2), is expressed specifically in the brain and spinal cord in Mammalia and Aves. We performed immunoblot analyses to elucidate the distribution of MHC-IIB (B2) in the brains of various animals. Nearly half of MHC-IIB existed as the B2 inserted isoform (MHC-IIB (B2)) in the cerebrum of the guinea-pig, rabbit and pig, while the non-B2 inse...

  2. Species Differences in the Distribution of the Nonmuscle Myosin Heavy Chain IIB Inserted Isoform in the Brain

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    Hagiwara, Shin-ya; Takahashi, Masayuki; Yamagishi, Akihiko

    2001-01-01

    The alternatively spliced isoform of the nonmuscle myosin heavy chain IIB (MHC-IIB) with an insert of 21 amino acids near the actin-binding region, MHC-IIB(B2), is expressed specifically in the brain and spinal cord in Mammalia and Aves. We performed immunoblot analyses to elucidate the distribution of MHC-IIB(B2) in the brains of various animals. Nearly half of MHC-IIB existed as the B2 inserted isoform (MHC-IIB(B2)) in the cerebrum of the guinea-pig, rabbit and pig, while the non-B2 inserte...

  3. Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: a ctenophore perspective

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    Dayraud Cyrielle

    2012-07-01

    Full Text Available Abstract Background Myosin II (or Myosin Heavy Chain II, MHCII is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa and striated muscle cells (MHCIIb. Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. Results We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus. Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene (PpiMHCIIa has an expression pattern akin to that of "stem cell markers" (Piwi, Vasa… and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. Conclusion MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue (PpiMHCIIb2 has retained MHCIIa-like expression features furthermore suggests that muscular

  4. Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

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    Van Dijk, J; Furch, M; Derancourt, J; Batra, R; Knetsch, M L; Manstein, D J; Chaussepied, P

    1999-03-01

    Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.

  5. Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development

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    Settleman Jeffrey

    2006-08-01

    Full Text Available Abstract Background The Rho-kinases (ROCKs are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity. Results We have examined a potential role for DRok in additional aspects of tissue morphogenesis using an activated form of the protein in transgenic flies. Our findings demonstrate that DRok activity can influence multiple morphogenetic processes, including eye and wing development. Furthermore, genetic studies reveal that Drok interacts with multiple downstream effectors of the Rho GTPase signaling pathway, including non-muscle myosin heavy chain, adducin, and Diaphanous in those developmental processes. Finally, in overexpression studies, we determined that Drok and Drosophila Lim-kinase interact in the developing nervous system. Conclusion These findings indicate widespread diverse roles for DRok in tissue morphogenesis during Drosophila development, in which multiple DRok substrates appear to be required.

  6. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9 and Rac1 activation

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    Mohammad G. Sabbir

    2016-04-01

    Full Text Available The Deleted in liver cancer 1 (Dlc1 gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9, plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype.

  7. Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

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    Hiller Gösta

    2009-06-01

    Full Text Available Abstract Background Deregulated activation of cyclin-dependent kinase-5 (Cdk5 is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems. Methods HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates. Results Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B. Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line. Conclusion A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.

  8. Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma.

    Science.gov (United States)

    Conti, Mary Anne; Saleh, Anthony D; Brinster, Lauren R; Cheng, Hui; Chen, Zhong; Cornelius, Shaleeka; Liu, Chengyu; Ma, Xuefei; Van Waes, Carter; Adelstein, Robert S

    2015-09-15

    To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (A(Nkx)/A(Nkx)) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 A(Nkx)/A(Nkx)) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In A(Nkx)/A(Nkx) mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

  9. Role of nonmuscle myosin IIB and N-RAP in cell spreading and myofibril assembly in primary mouse cardiomyocytes.

    Science.gov (United States)

    Lu, Shajia; Horowits, Robert

    2008-09-01

    We investigated the role of nonmuscle myosin heavy chain (NMHC) IIB in cultured embryonic mouse cardiomyocytes by specific knockdown using RNA interference. NMHC IIB protein levels decreased 90% compared with mock-transfected cells by 3 days post transfection. NMHC IIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP is a scaffold for alpha-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of alpha-actinin organization. Between 3 and 6 days, NMHC IIB knockdown was accompanied by the abolishment of cardiomyocyte spreading. During this period the rate of myofibril accumulation steadily decreased, correlating with the slowly decreasing levels of N-RAP. Between 6 and 8 days NMHC IIB and N-RAP protein levels recovered, and cardiomyocyte spreading and myofibril accumulation resumed. Inhibition of proteasome function using MG132 led to accumulation of excess N-RAP, and the secondary decrease in N-RAP that otherwise accompanied NMHC IIB knockdown was abolished. The results show that NMHC IIB knockdown led to decreased N-RAP levels through proteasome-mediated degradation. Furthermore, these proteins have distinct functional roles, with NMHC IIB playing a role in cardiomyocyte spreading and N-RAP functioning in myofibril assembly.

  10. Identification of an FHL1 protein complex containing gamma-actin and non-muscle myosin IIB by analysis of protein-protein interactions.

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    Lili Wang

    Full Text Available FHL1 is multifunctional and serves as a modular protein binding interface to mediate protein-protein interactions. In skeletal muscle, FHL1 is involved in sarcomere assembly, differentiation, growth, and biomechanical stress. Muscle abnormalities may play a major role in congenital clubfoot (CCF deformity during fetal development. Thus, identifying the interactions of FHL1 could provide important new insights into its functional role in both skeletal muscle development and CCF pathogenesis. Using proteins derived from rat L6GNR4 myoblastocytes, we detected FHL1 interacting proteins by immunoprecipitation. Samples were analyzed by liquid chromatography mass spectrometry (LC-MS. Dynamic gene expression of FHL1 was studied. Additionally, the expression of the possible interacting proteins gamma-actin and non-muscle myosin IIB, which were isolated from the lower limbs of E14, E15, E17, E18, E20 rat embryos or from adult skeletal muscle was analyzed. Potential interacting proteins isolated from E17 lower limbs were verified by immunoprecipitation, and co-localization in adult gastrocnemius muscle was visualized by fluorescence microscopy. FHL1 expression was associated with skeletal muscle differentiation. E17 was found to be the critical time-point for skeletal muscle differentiation in the lower limbs of rat embryos. We also identified gamma-actin and non-muscle myosin IIB as potential binding partners of FHL1, and both were expressed in adult skeletal muscle. We then demonstrated that FHL1 exists as part of a complex, which binds gamma-actin and non-muscle myosin IIB.

  11. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    Science.gov (United States)

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  12. Polymorphisms in the non-muscle myosin heavy chain gene (MYH9 are associated with lower glomerular filtration rate in mixed ancestry diabetic subjects from South Africa.

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    Tandi Edith Matsha

    Full Text Available OBJECTIVE: Though single nucleotide polymorphisms (SNPs in the non-muscle myosin gene (MYH9 have been reported to explain most of the excess risk of nondiabetic chronic kidney disease (CKD, in African-Americans, some studies have also shown associations with diabetic end-stage renal disease. We investigated the association of MYH9 SNPs with renal traits in a mixed-ancestry South African population prone to diabetes. RESEARCH DESIGN AND METHODS: Three SNPs known to be associated with CKD (rs4821480, rs5756152 and rs12107 were genotyped using Taqman assay in 716 adults (198 with diabetes from the Bellville-South community, Cape Town. Glomerular filtration rate was estimated (eGFR and urinary albumin/creatinine ratio (ACR assessed. Multivariable regressions were used to relate the SNPs with renal traits. RESULTS: Mean age was 53.6 years, with the expected differences observed in characteristics by diabetic status. Significant associations were found between rs575152 and serum creatinine, and eGFR in the total population, and in diabetic participants (all p≤0.003, but not in non-diabetics (all p≥0.16, with significant interactions by diabetes status (interaction-p≤0.009. The association with ACR was borderline in diabetic participants (p = 0.05 and non-significant in non-diabetics (p = 0.85, with significant interaction (interaction p = 0.02. rs12107 was associated with fasting-, 2-hour glucose and HbA1c in diabetic participants only (interaction-p≤0.003, but not with renal traits. CONCLUSION: MYH9 SNPs were associated with renal traits only in diabetic participants in this population. Our findings and other studies suggest that MYH9 may have a broader genetic risk effect on kidney diseases.

  13. Pilot study of an association between a common variant in the non-muscle myosin heavy chain 9 (MYH9) gene and type 2 diabetic nephropathy in a Taiwanese population

    OpenAIRE

    Hsieh, Chang-Hsun; Hung, Yi-Jen; Pei, Dee; Kuo, Shi-Wen; Lin, Eugene

    2010-01-01

    Nowadays diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD). Recent studies have demonstrated that the myosin, heavy chain 9, non-muscle (MYH9) gene is associated with ESRD in African Americans. In this study, we tested the hypothesis that a common single nucleotide polymorphism rs16996677 in the MYH9 gene may contribute to the etiology of DN in type 2 diabetes (T2D) in a Taiwanese population with T2D. There were 180 T2D patients diagnosed with DN and 178 age...

  14. Pilot study of an association between a common variant in the non-muscle myosin heavy chain 9 (MYH9 gene and type 2 diabetic nephropathy in a Taiwanese population

    Directory of Open Access Journals (Sweden)

    Chang-Hsun Hsieh

    2010-03-01

    Full Text Available Chang-Hsun Hsieh1, Yi-Jen Hung1, Dee Pei2, Shi-Wen Kuo3, Eugene Lin41Division of Endocrinology and Metabolism, Tri-Service General Hospital, Taipei; 2Division of Endocrinology and Metabolism, Cardinal Tien Hospital, Taipei County; 3Division of Endocrinology, Buddhist Xindian Tzu Chi General Hospital, Taipei; 4Vita Genomics Inc., Wugu Shiang, Taipei, TaiwanAbstract: Nowadays diabetic nephropathy (DN is the most common cause of end-stage renal disease (ESRD. Recent studies have demonstrated that the myosin, heavy chain 9, non-muscle (MYH9 gene is associated with ESRD in African Americans. In this study, we tested the hypothesis that a common single nucleotide polymorphism rs16996677 in the MYH9 gene may contribute to the etiology of DN in type 2 diabetes (T2D in a Taiwanese population with T2D. There were 180 T2D patients diagnosed with DN and 178 age- and sex-similar T2D without DN controls. Single locus analyses showed no significant main effects of MYH9 rs16996677 on the risk of DN in T2D. The results suggest that the rs16996677 SNP in MYH9 may not contribute to the risk of DN in T2D in Taiwanese T2D patients.Keywords: diabetic nephropathy, end-stage renal disease, single nucleotide polymorphisms, type 2 diabetes

  15. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

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    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  16. Non-muscle contractile proteins in the organ of corti

    Energy Technology Data Exchange (ETDEWEB)

    Thalmann, I.; Giometti, C.S.; Thalmann, R. (Washington Univ., St. Louis, MO (USA))

    1985-01-01

    Evidence indicates that an active contractile process exists in the outer hair cells of the mammalian cochlea. Proteins ordinarily associated with muscle contraction have been identified in the outer hair cells by immunohistologic techniques. On this basis a muscle-like mechanism of contraction/relaxation has been postulated by several investigators. The possibility must be considered, however, that the contractile proteins identified thus far in inner ear structures may be nonmuscle rather than muscle forms. In skeletal muscle, actin and myosin are responsible for the physical movement of the muscle fibers, and tropomyosin and troponin are involved in regulating this movement; these four proteins, as well as a variety of proteins involved with the normal cell maintenance functions are all of a muscle-specific type. Non-muscle-like motion also depends upon the interaction of actin with myosin; however, not only are these proteins structurally different from those specific to skeletal muscle but their proportions are also different. We have used two-dimensional polyacrylamide gel electrophoresis to study the proteins in freeze dried preparations of whole organ of Corti from the guinea pig. The identified proteins include non-muscle actin, three forms of non-muscle tropomyosin, alpha- and beta-tubulin, alpha-actinin, and lactate dehydrogenase (LDH B). Myosin heavy and light chains were not detected in the organ of Corti preparation, but the levels of those proteins might be too low to be detected with the protein load used of those proteins might be too low to be detected with the protein load used for this analysis. Although troponin could not be detected, calmodulin was present. All of these findings tend to indicate that the contraction/relaxation processes that have been associated with the organ of Corti by others are of the non-muscle variety.

  17. Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration

    Science.gov (United States)

    Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S.

    2016-11-01

    Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

  18. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad

    Science.gov (United States)

    Ono, Kanako; Ono, Shoichiro

    2016-01-01

    The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle–like contractile apparatuses, it has a striated muscle–like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  19. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad.

    Science.gov (United States)

    Ono, Kanako; Ono, Shoichiro

    2016-04-01

    The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle-like contractile apparatuses, it has a striated muscle-like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath.

  20. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    Science.gov (United States)

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  1. Nonmuscle invasive bladder cancer:a primer on immunotherapy

    Institute of Scientific and Technical Information of China (English)

    Mahir Maruf; Sam J. Brancato; Piyush K. Agarwal

    2016-01-01

    Intravesical Bacillus Calmette-Guérin (BCG) has long been the gold standard treatment of nonmuscle invasive bladder cancer. Recently, there has been an emergence of novel immunotherapeutic agents, which have shown promise in the treatment of urothelial cell carcinoma. These agents aim to augment, modify, or enhance the immune response. Such strategies include recombinant BCG, monoclonal antibodies, vaccines, gene therapy, and adoptive T-cell therapy. Here, we review the emerging immunotherapeutics in the treatment of nonmuscle invasive bladder cancer.

  2. Long-range self-organization of cytoskeletal myosin II filament stacks.

    Science.gov (United States)

    Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

    2017-02-01

    Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

  3. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

    OpenAIRE

    Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook

    2010-01-01

    Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology–pleckstrin homology modu...

  4. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  5. Myosin individualized: single nucleotide polymorphisms in energy transduction

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    Wieben Eric D

    2010-03-01

    Full Text Available Abstract Background Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC. Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. Results An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. Conclusions Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these

  6. Novel Simulation Model of Non-Muscle Invasive Bladder Cancer

    DEFF Research Database (Denmark)

    Patel, Sanjay R; Dinh, Tuan; Noah-Vanhoucke, Joyce

    2015-01-01

    Introduction: There have been no randomized controlled trials (RCTs) evaluating the clinical or economic benefit of mitomycin C intravesical therapy vs. radical cystectomy in patients with high-risk non-muscle invasive bladder cancer (NMIBC). We used the Archimedes computational model to simulate...

  7. At the Start of the Sarcomere: A Previously Unrecognized Role for Myosin Chaperones and Associated Proteins during Early Myofibrillogenesis

    Directory of Open Access Journals (Sweden)

    J. Layne Myhre

    2012-01-01

    Full Text Available The development of striated muscle in vertebrates requires the assembly of contractile myofibrils, consisting of highly ordered bundles of protein filaments. Myofibril formation occurs by the stepwise addition of complex proteins, a process that is mediated by a variety of molecular chaperones and quality control factors. Most notably, myosin of the thick filament requires specialized chaperone activity during late myofibrillogenesis, including that of Hsp90 and its cofactor, Unc45b. Unc45b has been proposed to act exclusively as an adaptor molecule, stabilizing interactions between Hsp90 and myosin; however, recent discoveries in zebrafish and C. elegans suggest the possibility of an earlier role for Unc45b during myofibrillogenesis. This role may involve functional control of nonmuscle myosins during the earliest stages of myogenesis, when premyofibril scaffolds are first formed from dynamic cytoskeletal actin. This paper will outline several lines of evidence that converge to build a model for Unc45b activity during early myofibrillogenesis.

  8. Model for processive movement of myosin Ⅴ and myosin

    Institute of Scientific and Technical Information of China (English)

    Xie Ping; Dou Shuo-Xing; Wang Peng-Ye

    2005-01-01

    Myosin Ⅴ and myosin Ⅵ are two classes of two-headed molecular motors of the myosin superfamily that move processively along helical actin filaments in opposite directions. Here we present a hand-over-hand model for their processive movements. In the model, the moving direction of a dimeric molecular motor is automatically determined by the relative orientation between its two heads at free state and its head's binding orientation on track filament.This determines that myosin Ⅴ moves toward the barbed end and myosin Ⅵ moves toward the pointed end of actin.During the moving period in one step, one head remains bound to actin for myosin Ⅴ whereas two heads are detached for myosin Ⅵ: the moving manner is determined by the length of neck domain. This naturally explains the similar dynamic behaviours but opposite moving directions of myosin Ⅵ and mutant myosin Ⅴ (the neck of which is truncated to only one-sixth of the native length). Because of different moving manners, myosin Ⅵ and mutant myosin Ⅴ exhibit significantly broader step-size distribution than native myosin Ⅴ. However, all the three motors give the same mean step size of ~36nm (the pseudo-repeat of actin helix). All these theoretical results are in agreement with previous experimental ones.

  9. Direct observation of motion of single F-actin filaments in the presence of myosin

    Science.gov (United States)

    Yanagida, Toshio; Nakase, Michiyuki; Nishiyama, Katsumi; Oosawa, Fumio

    1984-01-01

    Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems1-4, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell5. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.

  10. Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II-Dependent Cell Traction Force.

    Science.gov (United States)

    Hossain, M Moazzem; Zhao, Guangyi; Woo, Moon-Sook; Wang, James H-C; Jin, Jian-Ping

    2016-11-01

    Cell traction force (CTF) plays a critical role in controlling cell shape, permitting cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three calponin isoforms. Among them, calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts, and fibroblasts and plays a role in regulating cytoskeleton activities such as cell adhesion, migration, and cytokinesis. Knockout (KO) of the gene encoding calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of calponin 2 in modulating CTF. In this study, we examined fibroblasts isolated from Cnn2 KO and wild-type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured on polyacrylamide gel substrates embedded with fluorescent beads to measure root-mean-square traction, total strain energy, and net contractile movement. The results showed that calponin 2-null fibroblasts exhibit traction force greater than that of WT cells. Adherent calponin 2-null fibroblasts de-adhered faster than the WT control during mild trypsin treatment, consistent with an increased CTF. Blebbistatin, an inhibitor of myosin II ATPase, is more effective upon an alteration in cell morphology when calponin 2 is present in WT fibroblasts than that on Cnn2 KO cells, indicating their additive effects in inhibiting myosin motor activity. The novel finding that calponin 2 regulates myosin-dependent CTF in non-muscle cells demonstrates a mechanism for controlling cell motility-based functions.

  11. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

    Science.gov (United States)

    Beach, Jordan R; Bruun, Kyle S; Shao, Lin; Li, Dong; Swider, Zac; Remmert, Kirsten; Zhang, Yingfan; Conti, Mary A; Adelstein, Robert S; Rusan, Nasser M; Betzig, Eric; Hammer, John A

    2017-02-01

    The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

  12. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho

    2017-02-01

    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  13. A new role for myosin II in vesicle fission.

    Science.gov (United States)

    Flores, Juan A; Balseiro-Gomez, Santiago; Cabeza, Jose M; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

    2014-01-01

    An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.

  14. Various Themes of Myosin Regulation.

    Science.gov (United States)

    Heissler, Sarah M; Sellers, James R

    2016-05-01

    Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

  15. Phase 2 study of adjuvant intravesical instillations of apaziquone for high risk nonmuscle invasive bladder cancer.

    NARCIS (Netherlands)

    Hendricksen, K.; Cornel, E.B.; Reijke, T.M. de; Arentsen, H.C.; Chawla, S.; Witjes, J.A.

    2012-01-01

    PURPOSE: We studied the safety and efficacy of multiple adjuvant apaziquone instillations in patients with high risk nonmuscle invasive bladder cancer. MATERIALS AND METHODS: Patients with high risk nonmuscle invasive urothelial carcinoma of the bladder underwent transurethral resection of all bladd

  16. Specific Myosins Control Actin Organization, Cell Morphology, and Migration in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Makowska

    2015-12-01

    Full Text Available We investigated the myosin expression profile in prostate cancer cell lines and found that Myo1b, Myo9b, Myo10, and Myo18a were expressed at higher levels in cells with high metastatic potential. Moreover, Myo1b and Myo10 were expressed at higher levels in metastatic tumors. Using an siRNA-based approach, we found that knockdown of each myosin resulted in distinct phenotypes. Myo10 knockdown ablated filopodia and decreased 2D migration speed. Myo18a knockdown increased circumferential non-muscle myosin 2A-associated actin filament arrays in the lamella and reduced directional persistence of 2D migration. Myo9b knockdown increased stress fiber formation, decreased 2D migration speed, and increased directional persistence. Conversely, Myo1b knockdown increased numbers of stress fibers but did not affect 2D migration. In all cases, the cell spread area was increased and 3D migration potential was decreased. Therefore, myosins not only act as molecular motors but also directly influence actin organization and cell morphology, which can contribute to the metastatic phenotype.

  17. Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion.

    Directory of Open Access Journals (Sweden)

    Stephen J Terry

    Full Text Available Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.

  18. Emerging intravesical therapies for management of nonmuscle invasive bladder cancer

    Directory of Open Access Journals (Sweden)

    Jeffrey J Tomaszewski

    2010-05-01

    Full Text Available Jeffrey J Tomaszewski, Marc C SmaldoneDepartment of Urology, University of Pittsburgh School of Medicine, Pennsylvania, USAAbstract: Transitional cell carcinoma (TCC is the second most common urologic malignancy, and 70% of patients present with superficial or nonmuscle invasive bladder cancer (NMIBC. Intravesical bacillus Calmette-Guerin (BCG is the most effective agent for preventing disease recurrence, and the only therapy able to inhibit disease progression. However, recurrence rates as high as 30% and significant local and systemic toxicity have led to increased interest in alternative intravesical therapies. In patients refractory or intolerant to BCG, BCG-interferon α2b, gemcitabine, and anthracyclines (doxorubicin, epirubicin, valrubicin have demonstrated durable clinical responses. Phase I trials investigating alternative cytotoxic agents, such as apaziquone, taxanes (docetaxel, paclitaxel, and suramin are reporting promising data. Novel immunomodulating agents have demonstrated promise as efficacious alternatives in patients refractory to BCG. Optimization of existing chemotherapeutic regimens using hyperthermia, photodynamic therapy, magnetically-targeted carriers, and liposomes remains an area of active investigation. Despite enthusiasm for new intravesical agents, radical cystectomy remains the treatment of choice for patients with NMIBC who have failed intravesical therapy and selected patients with naïve T1 tumors and aggressive features. This report provides a comprehensive review of contemporary intravesical therapy for NMIBC and refractory NMIBC, with an emphasis on emerging agents and novel treatment modalities.Keywords: transitional cell carcinoma, nonmuscle, invasive, intravesical therapy, BCG

  19. Vanadate oligomer interactions with myosin.

    Science.gov (United States)

    Aureliano, M

    2000-05-30

    'Monovanadate' containing a mixture of at least four different vanadate species and 'decavanadate' containing apparently only two vanadate species, mainly decameric species, inhibit myosin and actomyosin ATPase activities. The addition of myosin to 'monovanadate' and 'decavanadate' solutions promotes differential increases on the 51V NMR spectral linewidths of vanadate oligomers. The relative order of line broadening upon myosin addition, reflecting the interaction of the vanadate oligomers with the protein, was V10 > V4 > V1 = 1, whereas no changes were observed for monomeric vanadate species. It is concluded that decameric and tetrameric vanadate species interact quite potently with the protein and affect myosin as well actomyosin ATPase activities.

  20. Mammalian Skeletal Muscle Fibres Promote Non-Muscle Stem Cells and Non-Stem Cells to Adopt Myogenic Characteristics

    Directory of Open Access Journals (Sweden)

    Taryn Morash

    2017-01-01

    Full Text Available Skeletal muscle fibres are unique cells in large animals, often composed of thousands of post-mitotic nuclei. Following skeletal muscle damage, resident stem cells, called satellite cells, commit to myogenic differentiation and migrate to carry out repair. Satellite stem cells migrate on muscle fibres through amoeboid movement, which relies on dynamic cell membrane extension and retraction (blebbing. It is not known whether blebbing is due to the intrinsic properties of satellite cells, or induced by features of the myofibre surface. Here, we determined the influence of the muscle fibre matrix on two important features of muscle regeneration: the ability to migrate and to differentiate down a myogenic lineage. We show that the muscle fibre is able to induce amoeboid movement in non-muscle stem cells and non-stem cells. Secondly, we show that prolonged co-culture on myofibres caused amniotic fluid stem cells and breast cancer cells to express MyoD, a key myogenic determinant. Finally, we show that amniotic fluid stem cells co-cultured on myofibres are able to fuse and make myotubes that express Myosin Heavy Chain.

  1. Anesthesia for trans-sternal thymectomy: modified non-muscle relaxant technique.

    Science.gov (United States)

    Baftiu, Nehat; Hadri, Burhan; Morina, Muharrem; Mustafa, Aziz

    2011-01-01

    Anesthesia for thymectomy in myasthenia gravis is challenging. Early surgical management is now considered to be an important therapeutic intervention for most of the patients of myasthenia gravis. The anesthetic experience of that technique is quite large. It involves either muscle relaxant or non-muscle relaxant techniques. However, the literature is deficient of standard anesthetic technique for thymectomy. Therefore we present in this report a modified non-muscle relaxant technique for thymectomy. We report one case with thymectomy under general anesthesia using fentanyl and propofol for induction and endotracheal intubation using non-muscle relaxant technique. The intubating, intraoperative and postoperative conditions were excellent.

  2. Natural biology and management of nonmuscle invasive bladder cancer

    DEFF Research Database (Denmark)

    Scarpato, Kristen R; Tyson, Mark D; Clark, Peter E

    2016-01-01

    PURPOSE OF REVIEW: This article reviews the natural biology of noninvasive bladder cancer and its management strategies while summarizing the most recent advances in the field. RECENT FINDINGS: Nonmuscle invasive bladder cancer (NMIBC) has a tendency to recur and progress. Risk stratification has...... treatment, especially in refractory high-risk cases, include the addition of intravesical hyperthermia, combination and sequential therapy with existing agents and the use of novel agents such as mycobacterial cell wall extract. New data are emerging regarding the potential role of active surveillance...... in low-risk patients. SUMMARY: NMIBC represents a variety of disease states and continues to pose management challenges. As our understanding of tumor biology improves and technology advances, achieving better outcomes through individualized care may be possible....

  3. Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site

    Energy Technology Data Exchange (ETDEWEB)

    Garabedian, T.E.; Yount, R.G. (Washington State Univ., Pullman (USA))

    1990-12-25

    The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with ({sup 3}H)UDP. ({sup 3}H) UDP was stably trapped at the active site by addition of vanadate (Vi) and Co{sup 2+}. The extraordinary stability of the myosin.Co2+.(3H)UDP.Vi complex (t1/2 greater than 5 days at 0{degrees}C) allowed it to be purified free of extraneous ({sup 3}H)UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped ({sup 3}H)UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results using ({sup 3}H)UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a zero-length cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by ({sup 3}H)UTP photolabeling in Acanthamoeba myosin II.

  4. Anesthesia for thoracoscopic thymectomy: modified non-muscle relaxant technique--case reports.

    Science.gov (United States)

    El-Dawlatly, Abdelazeem A

    2007-02-01

    Anesthesia for thymectomy in myasthenia gravis is challenging. The anesthetic experience of that technique is quite large. In involves either muscle relaxant or non-muscle relaxant techniques. However, the literature is deficient of standard anesthetic technique for thoracoscopic thymectomy. Therefore we present in this report a modified non-muscle relaxant technique for thoracoscopic thymectomy (TT). We report two cases who underwent TT under general anesthesia using sufentanil and propofol for induction and local anesthesia spray to the vocal cords to facilitate endobronchial intubation using non-muscle relaxant technique. The intubating, operating and postoperative conditions were excellent. To the best of our knowledge, this is the first report on modified non-muscle relaxant technique for TT in myasthenia gravis. Further cases have to be done to verify our technique.

  5. Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development.

    Directory of Open Access Journals (Sweden)

    Sokol V Todi

    Full Text Available BACKGROUND: Myosin VIIA (MyoVIIA is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck, are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase. Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research.

  6. The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract

    DEFF Research Database (Denmark)

    Hansen, Lars; Comyn, Sophie; Mang, Yuan;

    2014-01-01

    Genome-wide linkage analysis, followed by targeted deep sequencing, in a Danish multigeneration family with juvenile cataract revealed a region of chromosome 17 co-segregating with the disease trait. Affected individuals were heterozygous for two potentially protein-disrupting alleles in this reg......Genome-wide linkage analysis, followed by targeted deep sequencing, in a Danish multigeneration family with juvenile cataract revealed a region of chromosome 17 co-segregating with the disease trait. Affected individuals were heterozygous for two potentially protein-disrupting alleles......-type embryos resulted in development of a phenotype similar to the steif mutant. The p.Arg805Trp alteration in the mammalian UNC45B gene suggests that developmental cataract may be caused by a defect in non-muscle myosin assembly during maturation of the lens fiber cells.European Journal of Human Genetics...

  7. Obscurins: Goliaths and Davids take over non-muscle tissues.

    Science.gov (United States)

    Ackermann, Maegen A; Shriver, Marey; Perry, Nicole A; Hu, Li-Yen R; Kontrogianni-Konstantopoulos, Aikaterini

    2014-01-01

    Obscurins comprise a family of proteins originally identified in striated muscles, where they play essential roles in myofibrillogenesis, cytoskeletal organization, and Ca(2+) homeostasis. They are encoded by the single OBSCN gene, and are composed of tandem adhesion domains and signaling motifs. To date, two giant obscurin isoforms have been described in detail that differ only at the extreme COOH-terminus; while obscurin-A (∼720 kDa) contains a non-modular COOH-terminus that harbors binding sites for the adaptor proteins ankyrins, obscurin-B (∼870 kDa) contains two COOH-terminal serine-threonine kinase domains preceded by adhesion motifs. Besides the two known giant obscurins, a thorough search of transcript databases suggests that complex alternative splicing of the obscurin transcript results in the generation of additional giant as well as small isoforms with molecular masses ranging between ∼50-970 kDa. These novel isoforms share common domains with the characterized isoforms, but also contain unique regions. Using a panel of highly specific antibodies directed against epitopes spanning the entire length of giant obscurins, we employed western blotting and immunohistochemistry to perform a systematic and comprehensive characterization of the expression profile of obscurins in muscle and non-muscle tissues. Our studies demonstrate for the first time that obscurins are not restricted to striated muscles, but are abundantly expressed in several tissues and organs including brain, skin, kidney, liver, spleen, and lung. While some obscurin isoforms are ubiquitously expressed, others are preferentially present in specific tissues and organs. Moreover, obscurins are present in select structures and cell types where they assume nuclear, cytosolic, and membrane distributions. Given the ubiquitous expression of some obscurins, along with the preferential expression of others, it becomes apparent that obscurins may play common and unique roles, respectively, in

  8. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    Energy Technology Data Exchange (ETDEWEB)

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  9. Defining and treating the spectrum of intermediate risk nonmuscle invasive bladder cancer

    NARCIS (Netherlands)

    Kamat, A.M.; Witjes, J.A.; Brausi, M.; Soloway, M.; Lamm, D.; Persad, R.; Buckley, R.; Bohle, A.; Colombel, M.; Palou, J.

    2014-01-01

    PURPOSE: Low, intermediate and high risk categories have been defined to help guide the treatment of patients with nonmuscle invasive bladder cancer (Ta, T1, CIS). However, while low and high risk disease has been well classified, the intermediate risk category has traditionally comprised a heteroge

  10. Defining progression in nonmuscle invasive bladder cancer: it is time for a new, standard definition

    NARCIS (Netherlands)

    Lamm, D.; Persad, R.; Brausi, M.; Buckley, R.; Witjes, J.A.; Palou, J.; Bohle, A.; Kamat, A.M.; Colombel, M.; Soloway, M.

    2014-01-01

    PURPOSE: Despite being one of the most important clinical outcomes in nonmuscle invasive bladder cancer, there is currently no standard definition of disease progression. Major clinical trials and meta-analyses have used varying definitions or have failed to define this end point altogether. A stand

  11. Implementasi Program Pembinaan Napi di Lapas Kelas II B Metro

    Directory of Open Access Journals (Sweden)

    Narsidi Narsidi

    2004-01-01

    Full Text Available Penelitian ini bertujuan untuk mengungkap kesiapan sarana fisik dan nonfisik, jenis program pembinaan yang dilaksanakan, proses pelaksanaan program pembinaan, hasil, dan kemanfaatan program bagi narapidana di Lapas. Penelitian ini adalah jenis penelitian studi kasus dengan pendekatan kualitatif fenomenologis naturalistik. Pengumpulan data dilakukan melalui teknik observasi dan wawancara Teknik analisis data menggunakan model analisis interaktif dari Miles dan Huberman (1984. Hasil analisis data menunjukkan, bahwa Lapas Kelas II B Metro secara nonfisik masih memiliki kekurangan, baik kuantitas maupun kualitas. Proses pembinaan narapidana di Lapas belum banyak mengadopsi teori pembelajaran orang dewasa sehingga model pembelajarannya masih cenderung menggunakan model pembelajaran anak-anak (paedagogik. Secara umum dapat dikatakan bahwa pembinaan yang dilakukan Lapas dirasakan kemanfaatannya oleh narapidana.Namun, pembinaan yang dilakukan belum dapat memenuhi minat dan kebutuhan narapidana. Kata kunci: implementasi, program, pembinaan, narapidana.

  12. Myosin IIb activity and phosphorylation status determines dendritic spine and post-synaptic density morphology.

    Directory of Open Access Journals (Sweden)

    Jennifer L Hodges

    Full Text Available Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.

  13. The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract.

    Science.gov (United States)

    Hansen, Lars; Comyn, Sophie; Mang, Yuan; Lind-Thomsen, Allan; Myhre, Layne; Jean, Francesca; Eiberg, Hans; Tommerup, Niels; Rosenberg, Thomas; Pilgrim, David

    2014-11-01

    Genome-wide linkage analysis, followed by targeted deep sequencing, in a Danish multigeneration family with juvenile cataract revealed a region of chromosome 17 co-segregating with the disease trait. Affected individuals were heterozygous for two potentially protein-disrupting alleles in this region, in ACACA and UNC45B. As alterations of the UNC45B protein have been shown to affect eye development in model organisms, effort was focused on the heterozygous UNC45B missense mutation. UNC45B encodes a myosin-specific chaperone that, together with the general heat shock protein HSP90, is involved in myosin assembly. The mutation changes p.Arg805 to Trp in the UCS domain, an amino acid that is highly conserved from yeast to human. UNC45B is strongly expressed in the heart and skeletal muscle tissue, but here we show expression in human embryo eye and zebrafish lens. The zebrafish mutant steif, carrying an unc45b nonsense mutation, has smaller eyes than wild-type embryos and shows accumulation of nuclei in the lens. Injection of RNA encoding the human wild-type UNC45B protein into the steif homozygous embryo reduced the nuclei accumulation and injection of human mutant UNC45B cDNA in wild-type embryos resulted in development of a phenotype similar to the steif mutant. The p.Arg805Trp alteration in the mammalian UNC45B gene suggests that developmental cataract may be caused by a defect in non-muscle myosin assembly during maturation of the lens fiber cells.

  14. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

    Science.gov (United States)

    Ducret, Axel; Nguyen, Mai; Breckenridge, David G; Shore, Gordon C

    2003-01-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.

  15. Muscle myosin filaments: cores, crowns and couplings.

    Science.gov (United States)

    Squire, John M

    2009-09-01

    Myosin filaments in muscle, carrying the ATPase myosin heads that interact with actin filaments to produce force and movement, come in multiple varieties depending on species and functional need, but most are based on a common structural theme. The now successful journeys to solve the ultrastructures of many of these myosin filaments, at least at modest resolution, have not been without their false starts and erroneous sidetracks, but the picture now emerging is of both diversity in the rotational symmetries of different filaments and a degree of commonality in the way the myosin heads are organised in resting muscle. Some of the remaining differences may be associated with how the muscle is regulated. Several proteins in cardiac muscle myosin filaments can carry mutations associated with heart disease, so the elucidation of myosin filament structure to understand the effects of these mutations has a clear and topical clinical relevance.

  16. Myosin lever arm directs collective motion on cellular actin network.

    Science.gov (United States)

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  17. Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells

    Science.gov (United States)

    Baird, Michelle A.; Billington, Neil; Wang, Aibing; Adelstein, Robert S.; Sellers, James R.; Fischer, Robert S.; Waterman, Clare M.

    2017-01-01

    The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A “pulses” occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell–cell or cell–ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase– or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. PMID:27881665

  18. Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila

    National Research Council Canada - National Science Library

    Okumura, Takashi; Sasamura, Takeshi; Inatomi, Momoko; Hozumi, Shunya; Nakamura, Mitsutoshi; Hatori, Ryo; Taniguchi, Kiichiro; Nakazawa, Naotaka; Suzuki, Emiko; Maeda, Reo; Yamakawa, Tomoko; Matsuno, Kenji

    2015-01-01

    .... Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively...

  19. Mechanochemical model for myosin V.

    Science.gov (United States)

    Craig, Erin M; Linke, Heiner

    2009-10-27

    A rigorous numerical test of a hypothetical mechanism of a molecular motor should model explicitly the diffusive motion of the motor's degrees of freedom as well as the transition rates between the motor's chemical states. We present such a Brownian dynamics, mechanochemcial model of the coarse-grain structure of the dimeric, linear motor myosin V. Compared with run-length data, our model provides strong support for a proposed strain-controlled gating mechanism that enhances processivity. We demonstrate that the diffusion rate of a detached motor head during motor stepping is self-consistent with known kinetic rate constants and can explain the motor's key performance features, such as speed and stall force. We present illustrative and realistic animations of motor stepping in the presence of thermal noise. The quantitative success and illustrative power of this type of model suggest that it will be useful in testing our understanding of a range of biological and synthetic motors.

  20. Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue.

    Science.gov (United States)

    Aranjuez, George; Burtscher, Ashley; Sawant, Ketki; Majumder, Pralay; McDonald, Jocelyn A

    2016-06-15

    Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germ line-derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.

  1. Characterization of Amoeba proteus myosin VI immunoanalog.

    Science.gov (United States)

    Dominik, Magdalena; Kłopocka, Wanda; Pomorski, Paweł; Kocik, Elzbieta; Redowicz, Maria Jolanta

    2005-07-01

    Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

  2. Dictyostelium myosin bipolar thick filament formation: importance of charge and specific domains of the myosin rod.

    Directory of Open Access Journals (Sweden)

    Daniel Hostetter

    2004-11-01

    Full Text Available Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF structures when a green fluorescent protein (GFP "head" is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines.

  3. Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer.

    Science.gov (United States)

    Lindén, Mårten; Lind, Sara Bergström; Mayrhofer, Corina; Segersten, Ulrika; Wester, Kenneth; Lyutvinskiy, Yaroslav; Zubarev, Roman; Malmström, Per-Uno; Pettersson, Ulf

    2012-01-01

    Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (pblot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.

  4. Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction.

    Directory of Open Access Journals (Sweden)

    Xinhua Zhang

    Full Text Available Partial bladder outlet obstruction (PBOO, a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB and is associated with detrusor overactivity (DO. PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM and reports suggest varied BLEB efficacy for different SM myosin (SMM isoforms and/or SMM vs nonmuscle myosin (NMM. We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC alternatively spliced isoform SM-A (associated with tonic-type SM increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20% only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable

  5. Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.; (RPI); (IIT); (SDSU); (Vermont)

    2009-07-01

    The subfragment 2/light meromyosin 'hinge' region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.

  6. Chromosomal imbalance in the progression of high-risk non-muscle invasive bladder cancer

    Directory of Open Access Journals (Sweden)

    Ørntoft Torben

    2009-05-01

    Full Text Available Abstract Background Non-muscle invasive bladder neoplasms with invasion of the lamina propria (stage T1 or high grade of dysplasia are at "high risk" of progression to life-threatening cancer. However, the individual course is difficult to predict. Chromosomal instability (CI is associated with high tumor stage and grade, and possibly with the risk of progression. Methods To investigate the relationship between CI and subsequent disease progression, we performed a case-control-study of 125 patients with "high-risk" non-muscle invasive bladder neoplasms, 67 with later disease progression, and 58 with no progression. Selection criteria were conservative (non-radical resections and full prospective clinical follow-up (> 5 years. We investigated primary lesions in 59, and recurrent lesions in 66 cases. We used Affymetrix GeneChip® Mapping 10 K and 50 K SNP microarrays to evaluate genome wide chromosomal imbalance (loss-of-heterozygosity and DNA copy number changes in 48 representative tumors. DNA copy number changes of 15 key instability regions were further investigated using QPCR in 101 tumors (including 25 tumors also analysed on 50 K SNP microarrays. Results Chromosomal instability did not predict any higher risk of subsequent progression. Stage T1 and high-grade tumors had generally more unstable genomes than tumors of lower stage and grade (mostly non-primary tumors following a "high-risk" tumor. However, about 25% of the "high-risk" tumors had very few alterations. This was independent of subsequent progression. Recurrent lesions represent underlying field disease. A separate analysis of these lesions did neither reflect any difference in the risk of progression. Of specific chromosomal alterations, a possible association between loss of chromosome 8p11 and the risk of progression was found. However, the predictive value was limited by the heterogeneity of the changes. Conclusion Chromosomal instability (CI was associated with "high risk

  7. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  8. Plant-specific myosin XI, a molecular perspective

    Directory of Open Access Journals (Sweden)

    Motoki eTominaga

    2012-09-01

    Full Text Available In eukaryotic cells, organelle movement, positioning, and communications are critical for maintaining cellular functions and are highly regulated by intracellular trafficking. Directional movement of motor proteins along the cytoskeleton is one of the key regulators of such trafficking. Most plants have developed a unique actin–myosin system for intracellular trafficking. Although the composition of myosin motors in angiosperms is limited to plant-specific myosin classes VIII and XI, there are large families of myosins, especially in class XI, suggesting functional diversification among class XI members. However, the molecular properties and regulation of each myosin XI member remains unclear.To achieve a better understanding of the plant-specific actin–myosin system, the characterization of myosin XI members at the molecular level is essential. In the first half of this review, we summarize the molecular properties of tobacco 175-kDa myosin XI, and in the later half, we focus on myosin XI members in Arabidopsis thaliana.Through detailed comparison of the functional domains of these myosins with the functional domain of myosin V, we look for possible diversification in enzymatic and mechanical properties among myosin XI members concomitant with their regulation.

  9. Multiple endocrine neoplasia type II B with symptoms suggesting Hirschsprung's disease: a case report.

    Science.gov (United States)

    Mahaffey, S M; Martin, L W; McAdams, A J; Ryckman, F C; Torres, M

    1990-01-01

    A 3-year-old child was referred with a tentative diagnosis of Hirschsprung's disease because of life-long constipation and "megacolon" demonstrated radiographically. Our rectal biopsy revealed hyperganglionosis suggestive of multiple endocrine neoplasia (MEN) type II B. This, in addition to an elevated serum calcitonin level, prompted surgical removal of her thyroid, which appeared grossly normal but on sectioning, contained a medullary carcinoma in each lobe. She remains disease-free 5 years later. Gastrointestinal symptoms are a significant component of the MEN type II B syndrome, and often antedate the full phenotypic expression of the syndrome and the development of potentially lethal endocrine neoplasms. On the basis of this experience, it is recommended that MEN II B be included in the differential diagnosis of chronic constipation.

  10. Fluorescence cystoscopy in patients with non-muscle invasive bladder cancer

    Directory of Open Access Journals (Sweden)

    I. G. Rusakov

    2015-01-01

    Full Text Available The main challenge of treating non-muscle invasive bladder cancer is multifocal tumors. Current methods of diagnosis are failed to detect all superficial flat tumor lesions in bladder mucosa. The use of fluorescence imaging with 5-aminolevulinic acid (5-ALA allows to improve the sensibility of routine cystoscopy, but low specificity decreases its diagnostic accuracy. The method of fluorescence imaging combined with local fluorescence spectroscopy developed in P.A. Herzen MCRI has been shown to increase the specificity from 71% to 84%. Thus, local fluorescence spectroscopy in visible fluorescence of 5-ALA-induced protoporphyrin allows to perform guided biopsy and decrease the rate of diagnostic mistakes. 

  11. Optimal Treatment for Intermediate- and High-Risk, Nonmuscle-Invasive Bladder Cancer

    Directory of Open Access Journals (Sweden)

    A.P.M. van der Meijden

    2006-01-01

    Full Text Available According to clinical and pathological factors the prognosis of a patient with non-muscle invasive bladder tumors can be assessed. The prognosis is determined by the likelihood of recurrence(30-70% and/or progression to muscle invasive bladder cancer(1-15%.Trans urethral resection of bladder tumors remains the initial therapy but adjuvant intravesical instillations are necessary.All patients benefit from a single immediate post operative instillation with a chemotherapeutic agent and for low risk tumors this is the optimal therapy.Patients with intermediate and high risk tumors need more intravesical chemo-or immunotherapy. Chemotherapy reduces recurrences but not progression. Intravesical immunotherapy(BCG prevents or delays progression. Patients at high risk for progression may need upfront cystectomy.

  12. Quality-of-life survey for patients diagnosed with nonmuscle-invasive bladder cancer.

    Science.gov (United States)

    Abáigar-Pedraza, I; Megías-Garrigós, J; Sánchez-Payá, J

    2016-05-01

    To determine the reliability and validity of a quality-of-life survey for patients with nonmuscle-invasive bladder cancer. A total of 180 patients were included in the study. We developed a survey with 21 questions grouped into 5 areas. The patients filled in this survey and the Functional Assessment of Cancer Therapy - Bladder Cancer (FACT-BL) survey. To assess reliability, we calculated Cronbach's alpha coefficient and the kappa index. To determine criterion validity, we studied the association between the scores obtained from our survey and those from the FACT-BL survey using the Pearson correlation coefficient. To determine the construct validity (factorial and discriminatory), we performed a factor analysis, comparing it with Student's t-test for the scores obtained according to the tumour characteristics of reduced quality of life (e.g., malignancies located at the trigone of the bladder). Cronbach's alpha reliability coefficient was .83, and the kappa index varied between .7 and 1. For the association study between the new survey and the FACT-BL survey, we measured an r=.82 for the overall score and between r=.68 (disease) and r=.97 (sex life) in the various measures. In the factor analysis, we measured a Kaiser-Meyer-Olkin index of .77 and performed the Barlett test (P<.001). The comparison between the scores, in the presence or absence of certain tumour characteristics, has shown a reduced quality of life when those characteristics are present, which was statistically significant (P<.05) in the majority of cases. Our survey to measure the quality of life of patients with nonmuscle-invasive bladder cancer is reliable and valid. Copyright © 2015 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  13. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance

    Science.gov (United States)

    van Rooij, Eva; Quiat, Daniel; Johnson, Brett A.; Sutherland, Lillian B.; Qi, Xiaoxia; Richardson, James A.; Kelm, Robert J.; Olson, Eric N.

    2009-01-01

    Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related microRNAs (miRNAs) within their introns, which, in turn, control muscle myosin content, myofiber identity and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, co-expresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 are functionally redundant, and play a dominant role in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance. PMID:19922871

  14. MHC class II B diversity in blue tits : A preliminary study

    NARCIS (Netherlands)

    Rivero-de Aguilar, Juan; Schut, Elske; Merino, Santiago; Martinez, Javier; Komdeur, Jan; Westerdahl, Helena

    2013-01-01

    In this study, we partly characterize major histocompatibility complex (MHC) class II B in the blue tit (Cyanistes caeruleus). A total of 22 individuals from three different European locations: Spain, The Netherlands, and Sweden were screened for MHC allelic diversity. The MHC genes were investigate

  15. 30 CFR 57.22604 - Blasting from the surface (II-B mines).

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Blasting from the surface (II-B mines). 57.22604 Section 57.22604 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR... MINES Safety Standards for Methane in Metal and Nonmetal Mines Explosives § 57.22604 Blasting from...

  16. Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle CellsV⃞

    OpenAIRE

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K.; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-01-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B1...

  17. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    Science.gov (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  18. Striated Acto-Myosin Fibers Can Reorganize and Register in Response to Elastic Interactions with the Matrix

    Science.gov (United States)

    Friedrich, Benjamin M.; Buxboim, Amnon; Discher, Dennis E.; Safran, Samuel A.

    2011-01-01

    The remarkable striation of muscle has fascinated many for centuries. In developing muscle cells, as well as in many adherent, nonmuscle cell types, striated, stress fiberlike structures with sarcomere-periodicity tend to register: Based on several studies, neighboring, parallel fibers at the basal membrane of cultured cells establish registry of their respective periodic sarcomeric architecture, but, to our knowledge, the mechanism has not yet been identified. Here, we propose for cells plated on an elastic substrate or adhered to a neighboring cell, that acto-myosin contractility in striated fibers close to the basal membrane induces substrate strain that gives rise to an elastic interaction between neighboring striated fibers, which in turn favors interfiber registry. Our physical theory predicts a dependence of interfiber registry on externally controllable elastic properties of the substrate. In developing muscle cells, registry of striated fibers (premyofibrils and nascent myofibrils) has been suggested as one major pathway of myofibrillogenesis, where it precedes the fusion of neighboring fibers. This suggests a mechanical basis for the optimal myofibrillogenesis on muscle-mimetic elastic substrates that was recently observed by several groups in cultures of mouse-, human-, and chick-derived muscle cells. PMID:21641316

  19. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases.

    Science.gov (United States)

    Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook

    2010-08-23

    Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

  20. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    Science.gov (United States)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  1. Precise positioning of myosin VI on endocytic vesicles in vivo.

    Directory of Open Access Journals (Sweden)

    David Altman

    2007-08-01

    Full Text Available Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.

  2. Nuclei of non-muscle cells bind centrosome proteins upon fusion with differentiating myoblasts.

    Directory of Open Access Journals (Sweden)

    Xavier Fant

    Full Text Available BACKGROUND: In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown. METHODOLOGY: In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis. CONCLUSIONS: Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.

  3. Nonmuscle-invasive bladder cancer: what's changing and what has changed.

    Science.gov (United States)

    Manikandan, Ramanitharan; Rodriguez, Oscar; Parada, Rubén; Palou Redorta, Joan

    2017-02-03

    Nonmuscle-invasive bladder cancer (NMIBC) is a challenging disease to manage primarily due to its varied clinical course. The management of NMIBC has witnessed a widespread change with respect to its diagnosis and treatment. Although transurethral resection (TUR) and adjuvant bacillus Calmette-Guerin (BCG) stills remain the cornerstone, newer protocols has come into vogue to achieve optimal care. On the basis of a literature review, we aimed to establish 'what changes has already occurred and what is expected in the future' in NMIBC. A Medline search was performed to identify the published literature with respect to diagnosis, treatment and future perspectives on NMIBC. Particular emphasis was directed to determinants such as the quality of TUR and the newer modifications, Re-TUR, current status of newer macroscopic and microscopic imaging, role of urinary biomarkers, clinical, histologic and molecular predictors of high-risk disease, administration of intravesical agents, salvage therapy in BCG recurrence and the current best practice guidelines were analyzed. Optimal TUR, restaging in select group, incorporation of newer endoscopic imaging and judicious administration of intravesical chemo-immunotherapeutic agents can contribute to better patient care. Although there is a plethora of urinary markers, there is insufficient evidence for their use in isolation. The future probably lies in identification of genetic markers to determine disease recurrence, nonresponders to standard treatment and early institution of alternative/targeted therapy.

  4. Comparison of Guidelines on Non-Muscle Invasive Bladder Cancer (EAU, CUA, AUA, NCCN, NICE).

    Science.gov (United States)

    Power, Nicholas E; Izawa, Jonathan

    2016-01-07

    Non-muscle invasive bladder cancer (NMIBC) represents a considerably diverse patient group and the management of this complex disease is debatable. A number of panels from Europe and North America have convened on the topic and recently released guideline documents. The purpose was to compare and contrast the NMIBC guideline recommendations from the EAU (Europe), CUA (Canada), NCCN (United States), AUA (United States), and NICE (United Kingdom). All unabridged guideline documents were reviewed by the authors and comparisons were completed according to major topics in NMIBC. Despite a paucity of high level evidence regarding the majority of management topics in NMIBC, there was general agreement among the various guideline panels. Differences mainly centered on the categories of evidence synthesized and grades of recommendations. Each document offers a unique presentation of the available literature and guideline recommendation. The guidelines for NMIBC from the EAU, CUA, AUA, NCCN, and NICE provide considerable consensus regarding the management of this often difficult disease. Clinicians are encouraged to familiarize themselves with all of the guidelines in order to determine which style of presentation would be most useful to their current practice.

  5. Cargo binding activates myosin VIIA motor function in cells.

    Science.gov (United States)

    Sakai, Tsuyoshi; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2011-04-26

    Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

  6. Influence of the cardiac myosin hinge region on contractile activity.

    OpenAIRE

    Margossian, S S; Krueger, J W; Sellers, J R; Cuda, G; Caulfield, J B; Norton, P.; Slayter, H. S.

    1991-01-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myos...

  7. In vivo definition of cardiac myosin-binding protein C's critical interactions with myosin.

    Science.gov (United States)

    Bhuiyan, Md Shenuarin; McLendon, Patrick; James, Jeanne; Osinska, Hanna; Gulick, James; Bhandary, Bidur; Lorenz, John N; Robbins, Jeffrey

    2016-10-01

    Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

  8. Myosin Assembly, Maintenance and Degradation in Muscle: Role of the Chaperone UNC-45 in Myosin Thick Filament Dynamics

    Directory of Open Access Journals (Sweden)

    David B. Pilgrim

    2008-09-01

    Full Text Available Myofibrillogenesis in striated muscle cells requires a precise ordered pathway to assemble different proteins into a linear array of sarcomeres. The sarcomere relies on interdigitated thick and thin filaments to ensure muscle contraction, as well as properly folded and catalytically active myosin head. Achieving this organization requires a series of protein folding and assembly steps. The folding of the myosin head domain requires chaperone activity to attain its functional conformation. Folded or unfolded myosin can spontaneously assemble into short myosin filaments, but further assembly requires the short and incomplete myosin filaments to assemble into the developing thick filament. These longer filaments are then incorporated into the developing sarcomere of the muscle. Both myosin folding and assembly require factors to coordinate the formation of the thick filament in the sarcomere and these factors include chaperone molecules. Myosin folding and sarcomeric assembly requires association of classical chaperones as well as folding cofactors such as UNC-45. Recent research has suggested that UNC-45 is required beyond initial myosin head folding and may be directly or indirectly involved in different stages of myosin thick filament assembly, maintenance and degradation.

  9. Cargo recognition and cargo-mediated regulation of unconventional myosins.

    Science.gov (United States)

    Lu, Qing; Li, Jianchao; Zhang, Mingjie

    2014-10-21

    Organized motions are hallmarks of living organisms. Such motions range from collective cell movements during development and muscle contractions at the macroscopic scale all the way down to cellular cargo (e.g., various biomolecules and organelles) transportation and mechanoforce sensing at more microscopic scales. Energy required for these biological motions is almost invariably provided by cellular chemical fuels in the form of nucleotide triphosphate. Biological systems have designed a group of nanoscale engines, known as molecular motors, to convert cellular chemical fuels into mechanical energy. Molecular motors come in various forms including cytoskeleton motors (myosin, kinesin, and dynein), nucleic-acid-based motors, cellular membrane-based rotary motors, and so on. The main focus of this Account is one subfamily of actin filament-based motors called unconventional myosins (other than muscle myosin II, the remaining myosins are collectively referred to as unconventional myosins). In general, myosins can use ATP to fuel two types of mechanomotions: dynamic tethering actin filaments with various cellular compartments or structures and actin filament-based intracellular transport. In contrast to rich knowledge accumulated over many decades on ATP hydrolyzing motor heads and their interactions with actin filaments, how various myosins recognize their specific cargoes and whether and how cargoes can in return regulate functions of motors are less understood. Nonetheless, a series of biochemical and structural investigations in the past few years, including works from our own laboratory, begin to shed lights on these latter questions. Some myosins (e.g., myosin-VI) can function both as cellular transporters and as mechanical tethers. To function as a processive transporter, myosins need to form dimers or multimers. To be a mechanical tether, a monomeric myosin is sufficient. It has been shown for myosin-VI that its cellular cargo proteins can play critical roles

  10. Are we following the guidelines on non-muscle invasive bladder cancer?

    Directory of Open Access Journals (Sweden)

    Leonardo Oliveira Reis

    2016-02-01

    Full Text Available ABSTRACT Objectives To evaluate the clinical practice of non-muscle invasive bladder cancer (NMIBC treatment in Brazil in relation to international guidelines: Sociedade Brasileira de Urologia (SBU, European Association of Urology (EAU and American Urological Association (AUA. Materials and Methods Cross-sectional study using questionnaires about urological practice on treatment of NMIBC during the 32nd Brazilian Congress of Urology. A total of 650 question forms were answered. Results There were 73% of complete answers (total of 476 question forms. In total, 246 urologists (51.68% lived in the southeast region and 310 (65.13% treat 1 to 3 cases of NMIBC per month. Low risk cancer: Only 35 urologists (7.5% apply the single intravesical dose of immediate chemotherapy with Mitomicin C recommended by the above guidelines. Adjuvant therapy with BCG 2 to 4 weeks after TUR is used by 167 participants (35.1% and 271 urologists (56.9% use only TUR. High risk tumors: 397 urologists (83.4% use adjuvant therapy, 375 (78.8% use BCG 2 to 4 weeks after TUR, of which 306 (64.3% referred the use for at least one year. Intravesical chemotherapy with Mitomicin C (a controversial recommendation was used by 22 urologists (4.6%. BCG dose raised a lot of discrepancies. Induction doses of 40, 80 and 120mg were referred by 105 (22%, 193 (40.4% and 54 (11.3% respectively. Maintenance doses of 40, 80 and 120mg were referred by 190 (48.7%, 144 (37.0% and 32 (8.2% urologists, respectively. Schemes of administration were also varied and the one cited by SWOG protocol was the most used: 142 (29.8%. Conclusion SBU, EAU and AUA guidelines are partially respected by Brazilian urologists, particularly in low risk tumors. In high risk tumors, concordance rates are comparable to international data. Further studies are necessary to fully understand the reasons of such disagreement.

  11. Are we following the guidelines on non-muscle invasive bladder cancer?

    Science.gov (United States)

    Reis, Leonardo Oliveira; Moro, Juliano Cesar; Ribeiro, Luis Fernando Bastos; Voris, Brunno Raphael Iamashita; Sadi, Marcos Vinicius

    2016-01-01

    ABSTRACT Objectives To evaluate the clinical practice of non-muscle invasive bladder cancer (NMIBC) treatment in Brazil in relation to international guidelines: Sociedade Brasileira de Urologia (SBU), European Association of Urology (EAU) and American Urological Association (AUA). Materials and Methods Cross-sectional study using questionnaires about urological practice on treatment of NMIBC during the 32nd Brazilian Congress of Urology. A total of 650 question forms were answered. Results There were 73% of complete answers (total of 476 question forms). In total, 246 urologists (51.68%) lived in the southeast region and 310 (65.13%) treat 1 to 3 cases of NMIBC per month. Low risk cancer: Only 35 urologists (7.5%) apply the single intravesical dose of immediate chemotherapy with Mitomicin C recommended by the above guidelines. Adjuvant therapy with BCG 2 to 4 weeks after TUR is used by 167 participants (35.1%) and 271 urologists (56.9%) use only TUR. High risk tumors: 397 urologists (83.4%) use adjuvant therapy, 375 (78.8%) use BCG 2 to 4 weeks after TUR, of which 306 (64.3%) referred the use for at least one year. Intravesical chemotherapy with Mitomicin C (a controversial recommendation) was used by 22 urologists (4.6%). BCG dose raised a lot of discrepancies. Induction doses of 40, 80 and 120mg were referred by 105 (22%), 193 (40.4%) and 54 (11.3%) respectively. Maintenance doses of 40, 80 and 120mg were referred by 190 (48.7%), 144 (37.0%) and 32 (8.2%) urologists, respectively. Schemes of administration were also varied and the one cited by SWOG protocol was the most used: 142 (29.8%). Conclusion SBU, EAU and AUA guidelines are partially respected by Brazilian urologists, particularly in low risk tumors. In high risk tumors, concordance rates are comparable to international data. Further studies are necessary to fully understand the reasons of such disagreement. PMID:27136464

  12. An immunohistochemical detection of actin and myosin in the indigenous bacteria-adhering sites of microvillous columnar epithelial cells in Peyer's patches and intestinal villi in the rat jejunoileum.

    Science.gov (United States)

    Inamoto, Tetsurou; Namba, Makiko; Qi, Wang-Mei; Yamamoto, Kenkichi; Yokoo, Yuh; Miyata, Hidenori; Kawano, Junichi; Yokoyama, Toshifumi; Hoshi, Nobuhiko; Kitagawa, Hiroshi

    2008-11-01

    The mechanism of physical elimination of indigenous bacteria was ultrastructurally and immunohistochemically investigated in microvillous columnar epithelial cells of Peyer's patches and intestinal villi of the rat jejunoileum. From ultrastructural observation, the microfilaments accumulated to form several electron-dense layers beneath the bacteria adhering to the cell membrane, which was slightly invaginated in the epithelial cells of Peyer's patches and intestinal villi. As the microfilamentous layers were forming, the end portions of invaginations were deformed into a cone-shape and were finally collapsed. At the same time, the end portions of the adhered bacteria were also deformed into cone-shapes. The bacterial cells were moved back toward the invagination orifices with no morphological change in their inner structure. From immunohistochemical observation, beta-actin and nonmuscle-type myosin were detected at the thin layer just beneath the invaginated cell membrane. These findings suggest that indigenous bacteria which adhere to epithelial cells are removed by only a physical action of actin and myosin filaments, but are not killed. This bacterial cell removal system might lead to the establishment of a settlement of indigenous bacteria on host cells.

  13. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    Science.gov (United States)

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Force-producing ADP state of myosin bound to actin.

    Science.gov (United States)

    Wulf, Sarah F; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G; Pylypenko, Olena; Sweeney, H Lee; Houdusse, Anne M; Schröder, Rasmus R

    2016-03-29

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.

  15. The role of urine markers, white light cystoscopy and fluorescence cystoscopy in recurrence, progression and follow-up of non-muscle invasive bladder cancer

    NARCIS (Netherlands)

    Karaoglu, I.; Heijden, A.G. van der; Witjes, J.A.

    2014-01-01

    Non-muscle invasive bladder cancer (NMIBC) accounts for approximately 70 % of all bladder cancer cases and represents a heterogeneous pathological entity, characterized by a variable natural history and oncological outcome. The combination of cystoscopy and urine cytology is considered the gold stan

  16. Summary and Recommendations from the National Cancer Institute's Clinical Trials Planning Meeting on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer

    NARCIS (Netherlands)

    Lerner, S.P.; Bajorin, D.F.; Dinney, C.P.; Efstathiou, J.A.; Groshen, S.; Hahn, N.M.; Hansel, D.; Kwiatkowski, D.; O'Donnell, M.; Rosenberg, J.; Svatek, R.; Abrams, J.S.; Al-Ahmadie, H.; Apolo, A.B.; Bellmunt, J.; Callahan, M.; Cha, E.K.; Drake, C.; Jarow, J.; Kamat, A.; Kim, W.; Knowles, M.; Mann, B.; Marchionni, L.; McConkey, D.; McShane, L.; Ramirez, N.; Sharabi, A.; Sharpe, A.H.; Solit, D.; Tangen, C.M.; Amiri, A.T.; Allen, E. Van; West, P.J.; Witjes, J.A.; Quale, D.Z.

    2016-01-01

    The NCI Bladder Cancer Task Force convened a Clinical Trials Planning Meeting (CTPM) Workshop focused on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer (NMIBC). Meeting attendees included a broad and multi-disciplinary group of clinical and research stakeholders and included leaders from

  17. Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; technology transfer from microarrays to PCR

    DEFF Research Database (Denmark)

    Andersen, Lars Dyrskjøt; Reinert, Thomas; Novoradovsky, A;

    2012-01-01

    Background: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. Meth...

  18. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

    Energy Technology Data Exchange (ETDEWEB)

    Minoda, Hiroki [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Okabe, Tatsuhiro; Inayoshi, Yuhri [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganeishi, Tokyo184-8588 (Japan); Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032 (Japan); Katayama, Eisaku [Graduate School of Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Wakabayashi, Takeyuki [Department of Biosciences, School of Science and Engineering, Teikyo University, Utsunomiya, Tochigiken 320-8551 (Japan); Akimoto, Tsuyoshi [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan); Sugi, Haruo, E-mail: sugi@kyf.biglobe.ne.jp [Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173-8605 (Japan)

    2011-02-25

    Research highlights: {yields} We succeeded in recording structural changes of hydrated myosin cross-bridges. {yields} We succeeded in position-marking the cross-bridges with site-directed antibodies. {yields} We recorded cross-bridge movement at different regions in individual cross-bridge. {yields} The movement was smallest at the cross-bridge-subfragment two boundary. {yields} The results provide evidence for the cross-bridge lever arm mechanism. -- Abstract: Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

  19. Myosin and Actin Filaments in Muscle: Structures and Interactions.

    Science.gov (United States)

    Squire, John M; Paul, Danielle M; Morris, Edward P

    2017-01-01

    In the last decade, improvements in electron microscopy and image processing have permitted significantly higher resolutions to be achieved (sometimes <1 nm) when studying isolated actin and myosin filaments. In the case of actin filaments the changing structure when troponin binds calcium ions can be followed using electron microscopy and single particle analysis to reveal what happens on each of the seven non-equivalent pseudo-repeats of the tropomyosin α-helical coiled-coil. In the case of the known family of myosin filaments not only are the myosin head arrangements under relaxing conditions being defined, but the latest analysis, also using single particle methods, is starting to reveal the way that the α-helical coiled-coil myosin rods are packed to give the filament backbones.

  20. Axial disposition of myosin heads in isometrically contracting muscles.

    Science.gov (United States)

    Juanhuix, J; Bordas, J; Campmany, J; Svensson, A; Bassford, M L; Narayanan, T

    2001-03-01

    Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time.

  1. Actin filaments on myosin beds: The velocity distribution

    Science.gov (United States)

    Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

    1995-12-01

    In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

  2. Kinetic Adaptations of Myosins for Their Diverse Cellular Functions.

    Science.gov (United States)

    Heissler, Sarah M; Sellers, James R

    2016-08-01

    Members of the myosin superfamily are involved in all aspects of eukaryotic life. Their function ranges from the transport of organelles and cargos to the generation of membrane tension, and the contraction of muscle. The diversity of physiological functions is remarkable, given that all enzymatically active myosins follow a conserved mechanoenzymatic cycle in which the hydrolysis of ATP to ADP and inorganic phosphate is coupled to either actin-based transport or tethering of actin to defined cellular compartments. Kinetic capacities and limitations of a myosin are determined by the extent to which actin can accelerate the hydrolysis of ATP and the release of the hydrolysis products and are indispensably linked to its physiological tasks. This review focuses on kinetic competencies that - together with structural adaptations - result in myosins with unique mechanoenzymatic properties targeted to their diverse cellular functions.

  3. Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

    Directory of Open Access Journals (Sweden)

    Anabelle S. Cornachione

    2014-07-01

    Full Text Available Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

  4. Activation of Endothelial Pro-resolving Anti-Inflammatory Pathways by Circulating Microvesicles from Non-muscular Myosin Light Chain Kinase-Deficient Mice.

    Science.gov (United States)

    Gaceb, Abderahim; Vergori, Luisa; Martinez, M C; Andriantsitohaina, Ramaroson

    2016-01-01

    Microvesicles, small membrane vesicles released from cells, have beneficial and/or deleterious effects in sepsis. We previously reported that non-muscle myosin light chain kinase (nmMLCK) deletion protects mice against endotoxic shock by reducing inflammation. Here, we have evaluated the consequences of nmMLCK deletion on microvesicle phenotypes and their effects on mouse aortic endothelial cells in association with vascular inflammation and endothelial dysfunction during endotoxic shock induced by lipopolysaccharide in mice. Treatment with lipopolysaccharide induced an increase in levels of circulating microvesicles in wild type but not in nmMLCK-deficient mice. Microvesicles from nmMLCK-deficient mice (MVs(nmMLCK-/-)) prevented the inflammatory effects of lipopolysaccharide with concomitant increase of anti- inflammatory and reduction of pro-inflammatory secretome in mouse aortic endothelial cells. In addition, MVs(nmMLCK-/-) reduced the efficacy of lipopolysaccharide to increase aortic oxidative and nitrosative stresses as well as macrophage infiltration in the aorta. Moreover, MVs(nmMLCK-/-) prevented ex vivo endothelial dysfunction, vascular hyporeactivity, and in vivo overproduction of nitric oxide in heart and liver in response to lipopolysaccharide. Altogether, these findings provide evidence that nmMLCK deletion generates circulating microvesicles displaying protective effects by activating endothelial pro-resolving anti-inflammatory pathways allowing the effective down-regulation of oxidative and nitrative stresses associated with endotoxic shock. Thus, nmMLCK plays a pivotal role in susceptibility to sepsis via the control of cellular activation and release of circulating microvesicles.

  5. Minimal Mechanochemical Model for the Processivity of Myosin VI

    Science.gov (United States)

    Yang, Yubo; Lowe, Ian; Tehver, Riina

    2014-03-01

    Myosin VI is an ATPase responsible for force generation in cells. It dimerizes upon actin binding, and is proposed to walk along the actin filament. Single headed reaction mechanism of myosin VI is well understood but much of its walking mechanism remains unclear. We aim to construct a minimum model for the myosin VI walking mechanism and explore the minimal requirements for processivity. We constructed a kinetic model for the stepping mechanism of Myosin VI using minimum assumptions. The kinetics of the myosin VI dimer is modeled as a three state linear reaction network with reaction rates extracted from relevant experiments. The time limiting step in in-vitro experiments (low APT concentration) is the diffusion of detached head. In this process the myosin dimer is modeled as a tethered polymer with a flexible joint at the dimerization site. The relevance of this polymer model is checked with coarse-grained simulation. We found that the motor maintains processivity for a wide range of kinetic parameters, however long persistence length for the lever arm is crucial for processivity especially under resistive load.

  6. Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm-Tn chain.

    Science.gov (United States)

    Mijailovich, Srboljub M; Kayser-Herold, Oliver; Li, Xiaochuan; Griffiths, Hugh; Geeves, Michael A

    2012-12-01

    The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k(-I). The resulting equilibrium constant K(B) ≡ 1/K(I) varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.

  7. Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

    DEFF Research Database (Denmark)

    Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt

    2008-01-01

    as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple...... of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

  8. Status Report and Future Plans for the PEP-II B Factory

    CERN Document Server

    Fisher, A S

    2001-01-01

    The PEP-II B Factory at SLAC has been in operation with the BABAR detector since the summer of 1999. The peak currents and luminosity steadily increased through the end of the 2000 run on October 31. By that time, 0.75 A of electrons (the design current) routinely collided with 1.4 A of positrons in 657 bunches, to give a peak luminosity of 2.6E33 cm^{-2} s^{-1}. We delivered an integrated luminosity of up to 184/pb per day and 1033 per week; BABAR logged a total of 25/fb. Three days of machine development at the end of the run raised the peak luminosity to 3.29E33, exceeding the design goal of 3.0. BABAR then logged data for an hour, starting with a peak of 3.20. Next, we achieved the design value for positron current, 2.14 A, operating without collisions. The major limitation has been growth in the size of the positron beam in the low-energy ring (LER) due to electron clouds and multipacting. Since the arcs benefit from antechambers and a TiN coating with low secondary emission, our efforts have concentrate...

  9. Global fit analysis of myosin-5b motility reveals thermodynamics of Mg2+-sensitive acto-myosin-ADP states.

    Directory of Open Access Journals (Sweden)

    Igor Chizhov

    Full Text Available Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg(2+-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg(2+-ions. Free Mg(2+-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg(2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg(2+-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg(2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔH(Mg and ΔS(Mg of a fast equilibrium between magnesium free (AM·D and magnesium bound acto-myosin-ADP (AM· Mg(2+D states and the corresponding enthalpic barriers associated with ADP release (ΔH1(‡ and ΔH2(‡. The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.

  10. Calcium-regulated import of myosin IC into the nucleus.

    Science.gov (United States)

    Maly, Ivan V; Hofmann, Wilma A

    2016-06-01

    Myosin IC is a molecular motor involved in intracellular transport, cell motility, and transcription. Its mechanical properties are regulated by calcium via calmodulin binding, and its functions in the nucleus depend on import from the cytoplasm. The import has recently been shown to be mediated by the nuclear localization signal located within the calmodulin-binding domain. In the present paper, it is demonstrated that mutations in the calmodulin-binding sequence shift the intracellular distribution of myosin IC to the nucleus. The redistribution is displayed by isoform B, described originally as the "nuclear myosin," but is particularly pronounced with isoform C, the normally cytoplasmic isoform. Furthermore, experimental elevation of the intracellular calcium concentration induces a rapid import of myosin into the nucleus. The import is blocked by the importin β inhibitor importazole. These findings are consistent with a mechanism whereby calmodulin binding prevents recognition of the nuclear localization sequence by importin β, and the steric inhibition of import is released by cell signaling leading to the intracellular calcium elevation. The results establish a mechanistic connection between the calcium regulation of the motor function of myosin IC in the cytoplasm and the induction of its import into the nucleus. © 2016 Wiley Periodicals, Inc.

  11. Influence of the cardiac myosin hinge region on contractile activity.

    Science.gov (United States)

    Margossian, S S; Krueger, J W; Sellers, J R; Cuda, G; Caulfield, J B; Norton, P; Slayter, H S

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

  12. Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

    Science.gov (United States)

    Newman-Smith, Erin; Kourakis, Matthew J; Reeves, Wendy; Veeman, Michael; Smith, William C

    2015-04-13

    The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

  13. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.

    Science.gov (United States)

    Schott, Daniel H; Collins, Ruth N; Bretscher, Anthony

    2002-01-01

    Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

  14. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

    Directory of Open Access Journals (Sweden)

    Hanson Maureen R

    2007-02-01

    Full Text Available Abstract Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2, which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1, myosin XI-6 (At MYA2, myosin XI-8 (At XI-B, myosin XI-15 (At XI-I, myosin XI-16 (At XI-J and myosin XI-17 (At XI-K were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2, previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and

  15. Myosin domain evolution and the primary divergence of eukaryotes.

    Science.gov (United States)

    Richards, Thomas A; Cavalier-Smith, Thomas

    2005-08-25

    Eukaryotic cells have two contrasting cytoskeletal and ciliary organizations. The simplest involves a single cilium-bearing centriole, nucleating a cone of individual microtubules (probably ancestral for unikonts: animals, fungi, Choanozoa and Amoebozoa). In contrast, bikonts (plants, chromists and all other protozoa) were ancestrally biciliate with a younger anterior cilium, converted every cell cycle into a dissimilar posterior cilium and multiple ciliary roots of microtubule bands. Here we show by comparative genomic analysis that this fundamental cellular dichotomy also involves different myosin molecular motors. We found 37 different protein domain combinations, often lineage-specific, and many previously unidentified. The sequence phylogeny and taxonomic distribution of myosin domain combinations identified five innovations that strongly support unikont monophyly and the primary bikont/unikont bifurcation. We conclude that the eukaryotic cenancestor (last common ancestor) had a cilium, mitochondria, pseudopodia, and myosins with three contrasting domain combinations and putative functions.

  16. Preliminary research on myosin light chain kinase in rabbit liver

    Institute of Scientific and Technical Information of China (English)

    Bin Ren; Hua-Qing Zhu; Zhao-Feng Luo; Qing Zhou; Yuan Wang; Yu-Zhen Wang

    2001-01-01

    AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction (RT-PCR);the MLCK was obtained from rabbit liver, and its activity was analyzed by γ-32P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg-L-1, the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.

  17. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    Science.gov (United States)

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  18. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    Science.gov (United States)

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  19. Myosin IIA deficient cells migrate efficiently despite reduced traction forces at cell periphery

    Directory of Open Access Journals (Sweden)

    Melissa H. Jorrisch

    2013-02-01

    Cell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull the cell body forward. While previous studies have shown that myosin II deficient cells migrate efficiently, whether these cells exert traction forces during cell migration in the absence of the major contractile machinery is currently unknown. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoform (A and B, we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Myosin IIA and IIB localized preferentially to the leading edge where traction force was greatest, and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar actin organization and focal adhesions as wild-type cells. Interestingly, myosin IIA deficient cells migrated faster than wild-type or myosin IIB deficient cells on both a rigid surface and a pillar array, yet myosin IIA deficient cells exerted significantly less traction force at the leading edge than wild-type or myosin IIB deficient cells. These results suggest that, in the absence of myosin IIA mediated force-generating machinery, cells move with minimal traction forces at the cell periphery, thus demonstrating the remarkable ability of cells to adapt and migrate.

  20. Myosin IIA deficient cells migrate efficiently despite reduced traction forces at cell periphery.

    Science.gov (United States)

    Jorrisch, Melissa H; Shih, Wenting; Yamada, Soichiro

    2013-04-15

    Cell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull the cell body forward. While previous studies have shown that myosin II deficient cells migrate efficiently, whether these cells exert traction forces during cell migration in the absence of the major contractile machinery is currently unknown. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoform (A and B), we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Myosin IIA and IIB localized preferentially to the leading edge where traction force was greatest, and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar actin organization and focal adhesions as wild-type cells. Interestingly, myosin IIA deficient cells migrated faster than wild-type or myosin IIB deficient cells on both a rigid surface and a pillar array, yet myosin IIA deficient cells exerted significantly less traction force at the leading edge than wild-type or myosin IIB deficient cells. These results suggest that, in the absence of myosin IIA mediated force-generating machinery, cells move with minimal traction forces at the cell periphery, thus demonstrating the remarkable ability of cells to adapt and migrate.

  1. Internal Motility in Stiffening Actin-Myosin Networks

    CERN Document Server

    Uhde, J; Sackmann, E; Parmeggiani, A; Frey, E; Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin

    2003-01-01

    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.

  2. Detection of myosin immunoanalogue in the yeast Candida albicans.

    Science.gov (United States)

    Ghazali, M; Rodier, M H; el Moudni, B; Quellard, N; Jacquemin, J L

    1995-06-01

    Detection and localization of myosin immunoanalogue protein in the yeast Candida albicans were achieved by immunoblotting, indirect immunofluorescence assay, and immunoelectron microscopy. A polypeptide with an M(r) about 110,000, from cytosolic extract and insoluble fraction in the corresponding membrane pellet, was reacted with polyclonal and monoclonal antibodies raised against vertebrate muscle myosin. This protein was located by immunofluorescence and immunoelectron microscopy in the cell cortex along the plasmalemma, in the cytoplasm, and in the septum corresponding to bud scar region situated between the yeast-mother cell and the bud.

  3. Cohesive energy of zincblende (A$^{III}$ B$^{V}$ and A$^{II}$B$^{VI}$) structured solids

    Indian Academy of Sciences (India)

    A S Verma; B K Sarkar; V K Jindal

    2010-05-01

    In this paper we present an expression relating the cohesive energy (coh in kcal/mol) of A$^{III}$B$^{V}$ and A$^{II}$ B$^{VI}$ semiconductors with the product of ionic charges ($Z_{1}Z_{2}$) and nearest-neighbour distance d (Å). The cohesive energy values of these solids exhibit a linear relationship when plotted on a log–log scale against the nearest-neighbour distance (Å), but fall on different straight lines according to the ionic charge product of the solids. A good agreement has been found between the experimental and calculated values of the cohesive energy of A$^{III}$B$^{V}$ and A$^{II}$B$^{VI}$ semiconductors.

  4. Characterization and evolution of MHC class II B genes in Galápagos marine iguanas (Amblyrhynchus cristatus).

    Science.gov (United States)

    Glaberman, Scott; Moreno, Maria A; Caccone, Adalgisa

    2009-08-01

    Major histocompatibility complex (MHC) class II molecules play a key role in the adaptive immune system of vertebrates. Class II B genes appear to evolve in a very different manner in mammals and birds. Orthology is commonly observed among mammal loci, while genes tend to cluster phylogenetically within bird species. Here we present class II B data from a representative of another major group of amniotes, the squamates (i.e. lizards, snakes, amphisbaenians), with the ultimate goal of placing mammalian and avian MHC evolution into a broader context. In this study, eight class II B cDNA sequences were obtained from the Galápagos marine iguana (Amblyrhynchus cristatus) which were divided into five locus groups, Amcr-DAB1 through -DAB5, based on similarities along most of the coding and noncoding portions of the transcribed gene. All marine iguana sequences were monophyletic with respect to class II genes from other vertebrates indicating that they originated from a common ancestral locus after squamates split from other reptiles. The beta-1 domain, which is involved in antigen binding, exhibited signatures of positive selection as well as interlocus gene conversion in both long and short tracts-a pattern also observed in birds and fish, but not in mammals. On the other hand, the beta-2 domain was divergent between gene groups, which is characteristic of mammals. Based on these results, we preliminarily show that squamate class II B genes have been shaped by a unique blend of evolutionary forces that have been observed in differing degrees in other vertebrates.

  5. Influence of Trace Amount of Calponin on Smooth Muscle Myosin in Different States

    Institute of Scientific and Technical Information of China (English)

    Jing-Xian YANG; Xiao-Hua FENG; Ying ZHANG; Yuan LIN

    2004-01-01

    Calponin(CaP),a thin filament-associated protein,is thought to be involved in modulating smooth muscle contractile activity,but the role and mechanism keep unknown.In this study,trace amount of calponin(TAC)was found to obviously influence myosin in different states in Ca2+-independent manner,suggesting a high efficient interaction between TAC and myosin.In this assay,the lowest ratio of CaP vs.myosin was 1:10,000,with the concentration of CaP 10,000-fold lower than that used previously.Myosin phosphorylation,myosin Mg2+-ATPase activity and protein binding activity were detected to determine the effects of TAC on the myosin in different states.The amount of precipitated myosin that bound to TAC was used as the index to determine the interaction between myosin and TAC in binding assay.Our data showed that in the absence of actin,TAC significantly increased the precipitation of unphosphorylated myosin,Ca2+-dependently or independently phosphorylated myosin by MLCK,and stimulated the Mg2+-ATPase activities of these myosins slightly but significantly.However,no obvious change of precipitation of myosin phosphorylated by PKA was observed,indicating the relatively selective effect of TAC.In the presence of actin,the increase of myosin precipitations was abolished,and no obvious change of actin precipitations and actinactivated myosin Mg2+-ATPase activities were observed implicating the high efficiency of TAC on myosin being present in the absence of actin.Although we can not give conclusive comments to our results,we propose that the high efficiency of TAC-myosin interaction is present when actin is dissociated from myosin,even if CaP/myosin ratio is very low;this high efficient interaction can be abolished by actin.However,why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.

  6. Kinetic properties and small-molecule inhibition of human myosin-6

    Science.gov (United States)

    Heissler, Sarah M.; Selvadurai, Jayashankar; Bond, Lisa M.; Fedorov, Roman; Kendrick-Jones, John; Buss, Folma; Manstein, Dietmar J.

    2012-01-01

    Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion. PMID:22884421

  7. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

    Science.gov (United States)

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  8. Identification of C16orf74 as a marker of progression in primary non-muscle invasive bladder cancer.

    Directory of Open Access Journals (Sweden)

    Won Tae Kim

    Full Text Available PURPOSE: Methylation-induced silencing of PRSS3 has been shown to be significantly associated with invasive bladder cancer, and expression of the C16orf74 gene locus has been shown to correlate positively with PRSS3. The aim of the current study was to evaluate the relationship between C16orf74 expression level and progression in non-muscle invasive bladder cancer (NMIBC. MATERIALS AND METHODS: C16orf74 mRNA levels were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR analysis of 193 tumor specimens from patients with primary NMIBC. Expression data were analyzed in terms of clinical and experimental parameters. Kaplan-Meier curves and multivariate Cox regression models, respectively, were used to determine progression-free survival and to identify independent predictive parameters of progression. RESULTS: Analysis using Kaplan-Meier curves revealed prolonged progression-free survival of high-C16orf74-expressors as compared to low-expressors (p<0.001. Multivariate Cox regression analysis revealed that low C16orf74 mRNA expression levels are a significant risk factor for disease progression in patients with primary NMIBC (HR: 10.042, CI:2.699-37.360, p = 0.001. CONCLUSIONS: Decreased expression of C16orf74 correlates significantly with progression in primary NMIBC. C16orf74 expression level represents a potentially useful marker for predicting progression in primary NMIBC patients.

  9. Engineering controllable bidirectional molecular motors based on myosin.

    Science.gov (United States)

    Chen, Lu; Nakamura, Muneaki; Schindler, Tony D; Parker, David; Bryant, Zev

    2012-02-19

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.

  10. Myosin-I molecular motors at a glance.

    Science.gov (United States)

    McIntosh, Betsy B; Ostap, E Michael

    2016-07-15

    Myosin-I molecular motors are proposed to play various cellular roles related to membrane dynamics and trafficking. In this Cell Science at a Glance article and the accompanying poster, we review and illustrate the proposed cellular functions of metazoan myosin-I molecular motors by examining the structural, biochemical, mechanical and cell biological evidence for their proposed molecular roles. We highlight evidence for the roles of myosin-I isoforms in regulating membrane tension and actin architecture, powering plasma membrane and organelle deformation, participating in membrane trafficking, and functioning as a tension-sensitive dock or tether. Collectively, myosin-I motors have been implicated in increasingly complex cellular phenomena, yet how a single isoform accomplishes multiple types of molecular functions is still an active area of investigation. To fully understand the underlying physiology, it is now essential to piece together different approaches of biological investigation. This article will appeal to investigators who study immunology, metabolic diseases, endosomal trafficking, cell motility, cancer and kidney disease, and to those who are interested in how cellular membranes are coupled to the underlying actin cytoskeleton in a variety of different applications.

  11. Myosin light chain genes in the turkey (Meleagris gallopavo).

    Science.gov (United States)

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  12. Drebrin attenuates the interaction between actin and myosin-V.

    Science.gov (United States)

    Ishikawa, Ryoki; Katoh, Kaoru; Takahashi, Ayumi; Xie, Ce; Oseki, Koushi; Watanabe, Michitoshi; Igarashi, Michihiro; Nakamura, Akio; Kohama, Kazuhiro

    2007-07-27

    Drebrin-A is an actin-binding protein localized in the dendritic spines of mature neurons, and has been suggested to affect spine morphology [K. Hayashi, T. Shirao, Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons, J. Neurosci. 19 (1999) 3918-3925]. However, no biochemical analysis of drebrin-A has yet been reported. In this study, we purified drebrin-A using a bacterial expression system, and characterized it in vitro. Drebrin-A bound to actin filaments with a stoichiometry of one drebrin molecule to 5-6 actin molecules. Furthermore, drebrin-A decreased the Mg-ATPase activity of myosin V. In vitro motility assay revealed that the attachment of F-actin to glass surface coated with myosin-V was decreased by drebrin-A, but once F-actin attached to the surface, the sliding speed of F-actin was unaffected by the presence of drebrin A. These findings suggest that drebrin-A may affect spine dynamics, vesicle transport, and other myosin-V-driven motility in neurons through attenuating the interaction between actin and myosin-V.

  13. MyosinV controls PTEN function and neuronal cell size.

    Science.gov (United States)

    van Diepen, Michiel T; Parsons, Maddy; Downes, C Peter; Leslie, Nicholas R; Hindges, Robert; Eickholt, Britta J

    2009-10-01

    The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P(3) (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss, required PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.

  14. Structural insight into the UNC-45–myosin complex

    DEFF Research Database (Denmark)

    Fratev, Filip; Jonsdottir, Svava Osk; Pajeva, Ilza

    2013-01-01

    in silico methods. Initially, the human UNC-45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B–MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which...

  15. Globular tail of myosin-V is bound to vamp/synaptobrevin.

    Science.gov (United States)

    Ohyama, A; Komiya, Y; Igarashi, M

    2001-02-01

    VAMP/synaptobrevin is one of a number of v-SNAREs involved in vesicular fusion events in neurons. In a previous report, VAMP was shown to form a complex with synaptophysin and myosin V, a motor protein based on the F-actin, and that myosin V was then released from the complex in a Ca(2+)-dependent manner. Here, we found that VAMP alone is bound to myosin V in a Ca(2+)-independent manner, and determined that the globular tail domain of myosin V is its binding site. The syntaxin-VAMP-myosin V formed in the presence of Ca(2+)/calmodulin (CaM). In the absence of CaM, only syntaxin-VAMP, or VAMP-myosin V complex was formed. Our results suggest that VAMP acts as a myosin V receptor on the vesicles and regulates formation of the complex.

  16. Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking.

    Science.gov (United States)

    Li, Jianchao; Lu, Qing; Zhang, Mingjie

    2016-08-01

    Unconventional myosins are a superfamily of actin-based molecular motors playing diverse roles including cellular trafficking, mechanical supports, force sensing and transmission, etc. The variable neck and tail domains of unconventional myosins function to bind to specific cargoes including proteins and lipid vesicles and thus are largely responsible for the diverse cellular functions of myosins in vivo. In addition, the tail regions, together with their cognate cargoes, can regulate activities of the motor heads. This review outlines the advances made in recent years on cargo recognition and cargo binding-induced regulation of the activity of several unconventional myosins including myosin-I, V, VI and X in cellular trafficking. We approach this topic by describing a series of high-resolution structures of the neck and tail domains of these unconventional myosins either alone or in complex with their specific cargoes, and by discussing potential implications of these structural studies on cellular trafficking of these myosin motors.

  17. Myosin superfamily: The multi-functional and irreplaceable factors in spermatogenesis and testicular tumors.

    Science.gov (United States)

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-15

    Spermatogenesis is a fundamental process in sexual development and reproduction, in which the diploid spermatogonia transform into haploid mature spermatozoa. This process is under the regulation of multiple factors and pathway. Myosin has been implicated in various aspects during spermatogenesis. Myosins constitute a diverse superfamily of actin-based molecular motors that translocate along microfilament in an ATP-dependent manner, and six kinds of myosins have been proved that function during spermatogenesis. In mitosis and meiosis, myosins play an important role in spindle assembly and positioning, karyokinesis and cytokinesis. During spermiogenesis, myosins participate in acrosomal formation, nuclear morphogenesis, mitochondrial translocation and spermatid individualization. In this review, we summarize current understanding of the functions of myosin in spermatogenesis and some reproductive system diseases such as testicular tumors and prostate cancer, and discuss the roles of possible upstream molecules which regulate myosin in these processes.

  18. Mechanical characterization of one-headed myosin-V using optical tweezers.

    Directory of Open Access Journals (Sweden)

    Tomonobu M Watanabe

    Full Text Available Class V myosin (myosin-V is a cargo transporter that moves along an actin filament with large (approximately 36-nm successive steps. It consists of two heads that each includes a motor domain and a long (23 nm neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 k(BT from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.

  19. TGF-β1 Causes EMT by regulating N-Acetyl Glucosaminyl Transferases via Downregulation of Non Muscle Myosin II-A through JNK/P38/PI3K pathway in lung cancer.

    Science.gov (United States)

    Khan, Ghulam Jilany; Gao, Yingsheng; Gu, Ming; Wang, Lai; Khan, Sara; Naeem, Farah; Yousef, Bashir Alsiddig; Roy, Debmalya; Semukunzi, Herve; Yuan, Shengtao; Sun, Li

    2017-08-07

    Epithelial to mesenchymal transition (EMT) is a major determinant of cancer metastasis and is closely linked with TGF-β1. Intracellular proteins, including E. Cadherin, N. Cadherin and Vimentin are directly related to EMT that affect cell migration and adhesion; on the other hand, non muscle myosin (NM) has a central role in cytokinesis, migration and adhesion. We aimed to explore the association of EMT and metastasis with TGF-β1 through regulation of non-muscle myosin II-A (NMII-A) and its interaction with Hexosamine Biosynthesis Pathway (HBP). Protein expression changes were assessed by western blotting and immunofluorescent staining while transcription level changes were assessed by qRT-PCR. EMT was assessed by phenotypic analysis, wound healing, proliferation and transwell migration assay in vitro while in vivo studies were conducted in BALB/c nude mice for lung orthotopic and tail vein metastasis models. We demonstrated that regulation of JNK/ P38/PI3K by TGF-β1 led to down expression of NMII-A which promoted EMT and lung cancer metastasis. This down expression of NMII-A conversely upregulated the expression of Core 2 N-acetyl Glucosaminyl Transferase mucin type (C2GnT-M) and further facilitated up and down regulation of N-acetylglucosaminyltransferase (GnT) -V and -III respectively; moreover, NMII-A K.D cells showed 3 times more tendency to migrate towards brain in vivo. The study reports a novel pathway through which NMII-A negatively regulates EMT and metastasis via up regulation of C2GnT-M, GnT-V and down expression of GnT-III. These findings of lung cancer may further be required to study in other cancer types. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Directory of Open Access Journals (Sweden)

    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  1. Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites.

    Science.gov (United States)

    Cruz-Rivera, M; Reyes-Torres, A; Reynoso-Ducoing, O; Flisser, A; Ambrosio, J R

    2006-07-01

    Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.

  2. Mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Musil, L.S.; Frail, D.E.; Merlie, J.P. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-05-01

    Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with (35S)methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

  3. Circulating tumor cells detection has independent prognostic impact in high-risk non-muscle invasive bladder cancer.

    Science.gov (United States)

    Gazzaniga, Paola; de Berardinis, Ettore; Raimondi, Cristina; Gradilone, Angela; Busetto, Gian Maria; De Falco, Elena; Nicolazzo, Chiara; Giovannone, Riccardo; Gentile, Vincenzo; Cortesi, Enrico; Pantel, Klaus

    2014-10-15

    High-risk non-muscle invasive bladder cancer (NMIBC) progresses to metastatic disease in 10-15% of cases, suggesting that micrometastases may be present at first diagnosis. The prediction of risks of progression relies upon EORTC scoring systems, based on clinical and pathological parameters, which do not accurately identify which patients will progress. Aim of the study was to investigate whether the presence of CTC may improve prognostication in a large population of patients with Stage I bladder cancer who were all candidate to conservative surgery. A prospective single center trial was designed to correlate the presence of CTC to local recurrence and progression of disease in high-risk T1G3 bladder cancer. One hundred two patients were found eligible, all candidate to transurethral resection of the tumor followed by endovesical adjuvant immunotherapy with BCG. Median follow-up was 24.3 months (minimum-maximum: 4-36). The FDA-approved CellSearch System was used to enumerate CTC. Kaplan-Meier methods, log-rank test and multivariable Cox proportional hazard analysis was applied to establish the association of circulating tumor cells with time to first recurrence (TFR) and progression-free survival. CTC were detected in 20% of patients and predicted both decreased TFR (log-rank p < 0.001; multivariable adjusted hazard ratio [HR] 2.92 [95% confidence interval: 1.38-6.18], p = 0.005), and time to progression (log-rank p < 0.001; HR 7.17 [1.89-27.21], p = 0.004). The present findings provide evidence that CTC analyses can identify patients with Stage I bladder cancer who have already a systemic disease at diagnosis and might, therefore, potentially benefit from systemic treatment.

  4. [Transurethral en bloc resection of non-muscle invasive bladder cancer. What is the state of the art?].

    Science.gov (United States)

    Kramer, M W; Wolters, M; Abdelkawi, I F; Merseburger, A S; Nagele, U; Gross, A; Bach, T; Kuczyk, M A; Herrmann, T R W

    2012-06-01

    Bladder cancer of the urothelium is the second most common malignancy among urological tumors. In view of a worldwide aging population and the fact that increased incidence rates are associated with higher age, new socioeconomic challenges will appear. Even nowadays the treatment of bladder cancer bears the highest lifetime treatment costs per patient among all forms of cancer. In conjunction with higher comorbidity rates among older patients urologists are facing new challenges in the treatment and care of patients with bladder cancer. The standard treatment for non-muscle invasive bladder cancer (NMIBC) is monopolar transurethral resection using resection loops (TURB). Based on experience in the surgical treatment of benign prostatic hyperplasia, different concepts of en bloc resection of bladder tumors using alternative energy resources (e.g. holmium laser, thulium laser and the water-jet HybridKnife) have been developed. Goals of new treatment modalities are reduction of perioperative and postoperative comorbidities, better pathological work-up of the specimens and increased recurrence-free survival. Postulated advantages using laser devices are a more precise cutting line as well as better hemostasis. The evidential value of this review is limited due to the lack of randomized, prospective studies. However, there is a tendency towards a limitation of perioperative and postoperative morbidities as well as higher chance of well-preserved tissues for better pathohistological evaluation using en bloc resection methods. More studies with long-term follow-up periods and better randomization are needed to clarify whether en bloc strategies provide better long-term oncological survival.

  5. Rho kinase's role in myosin recruitment to the equatorial cortex of mitotic Drosophila S2 cells is for myosin regulatory light chain phosphorylation.

    Directory of Open Access Journals (Sweden)

    Sara O Dean

    Full Text Available BACKGROUND: Myosin II recruitment to the equatorial cortex is one of the earliest events in establishment of the cytokinetic contractile ring. In Drosophila S2 cells, we previously showed that myosin II is recruited to the furrow independently of F-actin, and that Rho1 and Rok are essential for this recruitment [1]. Rok phosphorylates several cellular proteins, including the myosin regulatory light chain (RLC. METHODOLOGY/PRINCIPAL FINDINGS: Here we express phosphorylation state mimic constructs of the RLC in S2 cells to examine the role of RLC phosphorylation involving Rok in the localization of myosin. Phosphorylation of the RLC is required for myosin localization to the equatorial cortex during mitosis, and the essential role of Rok in this localization and for cytokinesis is to maintain phosphorylation of the RLC. The ability to regulate the RLC phosphorylation state spatio-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis is not phosphorylation of the RLC. CONCLUSIONS/SIGNIFICANCE: We conclude that the Rho1 pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC.

  6. A panel of prognostic protein markers for progression in non-muscle invasive bladder cancer - a multicenter tissue microarray validation study

    DEFF Research Database (Denmark)

    Fristrup, Niels; Birkenkamp-Demtröder, Karin; Ulhøi, Benedicte Parm

    2012-01-01

    Ta and T1 urothelial carcinomas. Transcripts from the five genes encoding these proteins were previously included in gene expression signatures for outcome prediction for non-muscle invasive bladder cancer (NMIBC). As a training-set, we used primary NMIBC tissue-microarray specimens from a Danish...... cohort of 283 patients with long-term follow-up. For validation of the results we used three independent patient cohorts with long-term follow-up from Sweden, Spain, and Taiwan. In total 649 primary NMIBC tissue-microarray specimens from patients with long-term follow-up were used. Protein expression...

  7. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Energy Technology Data Exchange (ETDEWEB)

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  8. Covalent immobilization of myosin for in-vitro motility of actin

    Indian Academy of Sciences (India)

    Ellis Bagga; Sunita Kumari; Rajesh Kumar; Rakesh Kumar; R P Bajpai; Lalit M Bharadwaj

    2005-11-01

    The present study reports the covalent immobilization of myosin on glass surface and in-vitro motility of actin-myosin biomolecular motor. Myosin was immobilized on poly-L-lysine coated glass using heterobifunctional cross linker EDC and characterized by AFM. The in-vitro motility of actin was carried out on the immobilized myosin. It was observed that velocity of actin over myosin increases with increasing actin concentration (0.4-1.0 mg/ml) and was found in the range of 0.40-3.25 m/s. The motility of actin-myosin motor on artificial surfaces is of immense importance for developing nanodevices for healthcare and engineering applications.

  9. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

    Science.gov (United States)

    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-06

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

  10. The kinetics of bivalent metal ion dissociation from myosin subfragments.

    Science.gov (United States)

    Bennett, A J; Bagshaw, C R

    1986-01-01

    Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.

  11. Biomechanical Analysis of Cuboid Osteotomy Lateral Column Lengthening for Stage II B Adult-Acquired Flatfoot Deformity: A Cadaveric Study

    Directory of Open Access Journals (Sweden)

    Haichao Zhou

    2017-01-01

    Full Text Available Purpose. To investigate the effect of cuboid osteotomy lateral column lengthening (LCL for the correction of stage II B adult-acquired flatfoot deformity in cadaver. Methods. Six cadaver specimens were loaded to 350 N. Flatfoot models were established and each was evaluated radiographically and pedobarographically in the following conditions: (1 intact foot, (2 flatfoot, and (3 cuboid osteotomy LCL (2, 3, 4, and 5 mm. Results. Compared with the flatfoot model, the LCLs showed significant correction of talonavicular coverage on anteroposterior radiographs and talus-first metatarsal angle on both anteroposterior and lateral radiographs (p<.05. Compared with the intact foot, the above angles of the LCLs showed no significant difference except the 2 mm LCL. In terms of forefoot pressure, medial pressure of the 2 mm LCL (p=.044 and lateral pressure of the 3, 4, and 5 mm LCLs showed statistical differences (p<.05, but lateral pressure of the 3 mm LCL was not more than the intact foot as compared to the 4 and 5 mm LCLs, which was less than medial pressure. Conclusion. Cuboid osteotomy LCL procedure avoids damage to subtalar joint and has a good effect on correction of stage II B adult-acquired flatfoot deformity with a 3 mm lengthening in cadavers.

  12. Myosin VI regulates actin structure specialization through conserved cargo-binding domain sites.

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    Mamiko Isaji

    Full Text Available Actin structures are often stable, remaining unchanged in organization for the lifetime of a differentiated cell. Little is known about stable actin structure formation, organization, or maintenance. During Drosophila spermatid individualization, long-lived actin cones mediate cellular remodeling. Myosin VI is necessary for building the dense meshwork at the cones' fronts. We test several ideas for myosin VI's mechanism of action using domain deletions or site-specific mutations of myosin VI. The head (motor and globular tail (cargo-binding domains were both needed for localization at the cone front and dense meshwork formation. Several conserved partner-binding sites in the globular tail previously identified in vertebrate myosin VI were critical for function in cones. Localization and promotion of proper actin organization were separable properties of myosin VI. A vertebrate myosin VI was able to localize and function, indicating that functional properties are conserved. Our data eliminate several models for myosin VI's mechanism of action and suggest its role is controlling organization and action of actin assembly regulators through interactions at conserved sites. The Drosophila orthologues of interaction partners previously identified for vertebrate myosin VI are likely not required, indicating novel partners mediate this effect. These data demonstrate that generating an organized and functional actin structure in this cell requires multiple activities coordinated by myosin VI.

  13. Evolutionary traces decode molecular mechanism behind fast pace of myosin XI

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    Syamaladevi Divya P

    2011-09-01

    Full Text Available Abstract Background Cytoplasmic class XI myosins are the fastest processive motors known. This class functions in high-velocity cytoplasmic streaming in various plant cells from algae to angiosperms. The velocities at which they process are ten times faster than its closest class V homologues. Results To provide sequence determinants and structural rationale for the molecular mechanism of this fast pace myosin, we have compared the sequences from myosin class V and XI through Evolutionary Trace (ET analysis. The current study identifies class-specific residues of myosin XI spread over the actin binding site, ATP binding site and light chain binding neck region. Sequences for ET analysis were accumulated from six plant genomes, using literature based text search and sequence searches, followed by triple validation viz. CDD search, string-based searches and phylogenetic clustering. We have identified nine myosin XI genes in sorghum and seven in grape by sequence searches. Both the plants possess one gene product each belonging to myosin type VIII as well. During this process, we have re-defined the gene boundaries for three sorghum myosin XI genes using fgenesh program. Conclusion Molecular modelling and subsequent analysis of putative interactions involving these class-specific residues suggest a structural basis for the molecular mechanism behind high velocity of plant myosin XI. We propose a model of a more flexible switch I region that contributes to faster ADP release leading to high velocity movement of the algal myosin XI.

  14. Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions.

    Science.gov (United States)

    Ketschek, Andrea R; Jones, Steven L; Gallo, Gianluca

    2007-09-01

    Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.

  15. Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells.

    Science.gov (United States)

    Paves, H; Truve, E

    2007-01-01

    Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation.

  16. Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction

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    Campbell Shelagh

    2011-07-01

    Full Text Available Abstract Background Myosin VI, encoded by jaguar (jar in Drosophila melanogaster, is a unique member of the myosin superfamily of actin-based motor proteins. Myosin VI is the only myosin known to move towards the minus or pointed ends of actin filaments. Although Myosin VI has been implicated in numerous cellular processes as both an anchor and a transporter, little is known about the role of Myosin VI in the nervous system. We previously recovered jar in a screen for genes that modify neuromuscular junction (NMJ development and here we report on the genetic analysis of Myosin VI in synaptic development and function using loss of function jar alleles. Results Our experiments on Drosophila third instar larvae revealed decreased locomotor activity, a decrease in NMJ length, a reduction in synaptic bouton number, and altered synaptic vesicle localization in jar mutants. Furthermore, our studies of synaptic transmission revealed alterations in both basal synaptic transmission and short-term plasticity at the jar mutant neuromuscular synapse. Conclusions Altogether these findings indicate that Myosin VI is important for proper synaptic function and morphology. Myosin VI may be functioning as an anchor to tether vesicles to the bouton periphery and, thereby, participating in the regulation of synaptic vesicle mobilization during synaptic transmission.

  17. Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation

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    Nucciotti, V.; Stringari, C.; Sacconi, L.; Vanzi, F.; Linari, M.; Piazzesi, G.; Lombardi, V.; Pavone, F. S.

    2009-02-01

    Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

  18. Microscopic model of the actin-myosin interaction in muscular contractions

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    Gaveau, B.; Moreau, M.; Schuman, B.

    2004-01-01

    We define and study a detailed many body model for the muscular contraction taking into account the various myosin heads. The state of the system is defined by the position of the actin and by an internal coordinate of rotation for each myosin head. We write a system of Fokker-Planck equations and calculate the average for the position, the number of attached myosin heads, and the total force exerted on the actin. We also study the correlation between these quantities, in particular between the number of attached myosin heads and the force on the actin.

  19. Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus

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    L.S. Brinn

    2010-09-01

    Full Text Available Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

  20. Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

    Science.gov (United States)

    Kensler, Robert W.; Craig, Roger; Moss, Richard L.

    2017-01-01

    Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium. PMID:28167762

  1. A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells.

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    Ronna Hertzano

    2008-10-01

    Full Text Available Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.

  2. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    Science.gov (United States)

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  3. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation.

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    Sam Walcott

    2015-11-01

    Full Text Available Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies.

  4. Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

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    C.C.R. Souza

    2013-02-01

    Full Text Available Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL. In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

  5. Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family

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    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2011-01-01

    The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the “DIL” domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL–YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs. PMID:22645548

  6. Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila.

    Science.gov (United States)

    Okumura, Takashi; Sasamura, Takeshi; Inatomi, Momoko; Hozumi, Shunya; Nakamura, Mitsutoshi; Hatori, Ryo; Taniguchi, Kiichiro; Nakazawa, Naotaka; Suzuki, Emiko; Maeda, Reo; Yamakawa, Tomoko; Matsuno, Kenji

    2015-04-01

    The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes. Copyright © 2015 by the Genetics Society of America.

  7. [Clinical relevance of myosin isoforms in the diaphragm].

    Science.gov (United States)

    Gayan-Ramirez, G; Decramer, M

    2000-06-01

    The diaphragm as a striated muscle is characterized by the repetition of a single element arranged in series: the sarcomere containing two kinds of myofilaments: a thick one constituted by the myosin, and a thin one primarily composed of actin. The myosin molecule consists of two heads where two myosin heavy chains (MHC) are fixed, a flexible hinge with two light (MLC) chains, and long rod-shaped tails. The diaphragm contains 4 MHC isoforms (MHC-slow, MHC-2A, MHC-2B, MHC-2X) and 6 MLC isoforms (MLC-1f, MLC-3f, MLC-1sa, MLC-1sb, MLC-2f, MLC-2s/v). In humans, the diaphragm contains mainly fibers expressing the isoforms MHC-slow, MHC-2A, and MLC-2f, MLC-2s et MLC-1f. For the mechanical properties of the different isoforms, there is a gradient from the MHC-slow to the MHC-2A, MHC-2B and MHC-2X/2B. According to the circumstances, the diaphragm will adapt towards a slow profile (COPD, cardiac failure and in animals: Duchenne muscular dystrophy, denervation-1 week, age-female, corticosteroids, chronic stimulation), or a fast profile (in animals: chronic hypoxia, denervation-2 weeks, age-males) or a more oxidative profile (in animals: cachexia, obesity). The reasons why the diaphragm adapts towards a slower or a faster muscle are not known. In fact, for a given pathological situation, several factors are able to influence the fiber composition of the diaphragm. Therefore, the net result of the influence of these different factors in terms of MHC and MLC diaphragm adaptation is difficult to predict.

  8. A role for myosin VI in the localization of axonal proteins.

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    Tommy L Lewis

    2011-03-01

    Full Text Available In neurons polarized trafficking of vesicle-bound membrane proteins gives rise to the distinct molecular composition and functional properties of axons and dendrites. Despite their central role in shaping neuronal form and function, surprisingly little is known about the molecular processes that mediate polarized targeting of neuronal proteins. Recently, the plus-end-directed motor Myosin Va was shown to play a critical role in targeting of transmembrane proteins to dendrites; however, the role of myosin motors in axonal targeting is unknown. Here we show that Myosin VI, a minus-end-directed motor, plays a vital role in the enrichment of proteins on the surface of axons. Engineering non-neuronal proteins to interact with Myosin VI causes them to become highly concentrated at the axonal surface in dissociated rat cortical neurons. Furthermore, disruption of either Myosin VI function or expression leads to aberrant dendritic localization of axonal proteins. Myosin VI mediates the enrichment of proteins on the axonal surface at least in part by stimulating dendrite-specific endocytosis, a mechanism that has been shown to underlie the localization of many axonal proteins. In addition, a version of Channelrhodopsin 2 that was engineered to bind to Myosin VI is concentrated at the surface of the axon of cortical neurons in mice in vivo, suggesting that it could be a useful tool for probing circuit structure and function. Together, our results indicate that myosins help shape the polarized distributions of both axonal and dendritic proteins.

  9. Myosin Ⅷ Regulates Protonemal Patterning and Developmental Timing in the Moss Physcomitrella patens

    Institute of Scientific and Technical Information of China (English)

    Shu-Zon Wua; Julie A. Ritchie; Ai-Hong Pan; Ralph S. Quatrano; Magdalena Bezanilla

    2011-01-01

    Plants have two classes of myosins.While recent work has focused on class Ⅺ myosins showing that myosin Ⅺ is responsible for organelle motility and cytoplasmic streaming,much less is known about the role of myosin Ⅷ in plant growth and development.We have used a combination of RNAi and insertional knockouts to probe myosin Ⅷ function in the moss Physcomitrella patens.We isolated △myo8ABCDE plants demonstrating that myosin Ⅷ is not required for plant viability.However,myosin Ⅷ mutants are smaller than wild-type plants in part due to a defect in cell size.Additionally,△myo8ABCDE plants produce more side branches and form gametophores much earlier than wild-type plants.In the absence of nutrient media,△myo8ABCDE plants exhibit significant protonemal patterning defects,including highly curved protonemal filaments,morphologically defective side branches,as well as an increase in the number of branches.Exogenous auxin partially rescues protonemal defects in △myo8ABCDE plants grown in the absence of nutrients.This result,together with defects in protonemal branching,smaller caulonemal cells,and accelerated development in the △myo8ABCDE plants,suggests that myosin Ⅷ is involved in hormone homeostasis in P patens.

  10. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.

    Science.gov (United States)

    Schwab, Ryan S; Ihnatovych, Ivanna; Yunus, Sharifah Z S A; Domaradzki, Tera; Hofmann, Wilma A

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms.

  11. My oh my(osin): Insights into how auditory hair cells count, measure, and shape.

    Science.gov (United States)

    Pollock, Lana M; Chou, Shih-Wei; McDermott, Brian M

    2016-01-18

    The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle's morphology and hearing.

  12. Modeling Hand-Over-Hand and Inchworm Steps in Myosin VI

    Science.gov (United States)

    Jack, Amanda; Lowe, Ian; Tehver, Riina

    Myosin VI is a molecular motor protein that moves along actin filaments to transport cargo within a cell. There is much experimental evidence that the myosin VI dimer moves ``hand-over-hand'' along actin; however, recent experiments suggest that the protein can also move via an ``inchworm'' mechanism. We created a mechanochemical kinetic model to predict myosin VI's behavior under different ATP, ADP, and force conditions, taking these alternative mechanisms into account. Our model's calculations agree well with experimental results and can also be used to predict myosin VI's behavior outside experimentally tested regimes, such as under forward force. We also predict an optimized motor function for the protein around physiological (-2 pN) load and anchoring under -3 pN load. By using our model to predict myosin VI's response to environmental change, we can gain insight into the behavior of a protein that can be difficult to observe experimentally.

  13. Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function.

    Science.gov (United States)

    Tiago, Teresa; Palma, Pedro S; Gutierrez-Merino, Carlos; Aureliano, Manuel

    2010-11-01

    Abstract The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.

  14. Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species.

    Science.gov (United States)

    Ayres Sá, L; Menezes, M A; dos Santos Mermelstein, C

    2001-08-01

    Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus. Copyright 2001 Wiley-Liss, Inc.

  15. Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

    Science.gov (United States)

    Tang, Wanjian; Blair, Cheavar A.; Walton, Shane D.; Málnási-Csizmadia, András; Campbell, Kenneth S.; Yengo, Christopher M.

    2017-01-01

    Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation. PMID:28119616

  16. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  17. Efficacy and safety of photodynamic therapy for recurrent, high grade nonmuscle invasive bladder cancer refractory or intolerant to bacille Calmette-Guérin immunotherapy.

    Science.gov (United States)

    Lee, Joo Yong; Diaz, Richilda Red; Cho, Kang Su; Lim, Meng Shi; Chung, Jae Seung; Kim, Won Tae; Ham, Won Sik; Choi, Young Deuk

    2013-10-01

    We evaluated the effectiveness of photodynamic therapy using Radachlorin in patients with high grade, nonmuscle invasive bladder cancer refractory or intolerant to bacillus Calmette-Guérin therapy who refused radical cystectomy. Between July 2009 and December 2011 photodynamic therapy was performed in 22 men and 12 women. Radachlorin (0.5 to 0.6 mg/kg) was injected intravenously 2 to 3 hours before photodynamic therapy. After complete transurethral resection, a diffuser using a 22Fr cystoscope was placed in the bladder for irradiation with a 662 nm laser. Output beam power was adjusted to 1.8 W and the light dose was 15 J/cm(2). Photodynamic therapy was performed for 16 to 30 minutes. Recurrence after photodynamic therapy was followed by regular cystoscopy at 1, 2 and 3 months, and at 3-month intervals thereafter for up to 2.8 years. Efficacy was assessed by cystoscopy, cytology and histology, and defined as the number of patients who were tumor free after initial photodynamic therapy. Mean ± SD patient age was 62.94 ± 8.71 years. Average followup was 26.74 ± 6.34 months (median 28.12). As the primary efficacy outcome, the recurrence-free rate was 90.9% at 12 months, 64.4% at 24 months and 60.1% at 30 months. As the secondary efficacy outcome, there was no statistical difference in mass size, carcinoma in situ, number of previous bacillus Calmette-Guérin administrations, number of transurethral bladder resections or tumor multiplicity on Kaplan-Meier analysis (each p >0.05). No evidence of severe adverse effects was detected after photodynamic therapy. Photodynamic therapy with Radachlorin is a safe, effective treatment for nonmuscle invasive bladder cancer refractory or intolerant to bacillus Calmette-Guérin therapy in select patients. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  18. Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis.

    Science.gov (United States)

    Boyne, J R; Yosuf, H M; Bieganowski, P; Brenner, C; Price, C

    2000-12-01

    MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G(1) and the growing bud tip during S and G(2) phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.

  19. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding.

    Science.gov (United States)

    Arden, Susan D; Tumbarello, David A; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-10-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

  20. Approaches to myosin modelling in a two-phase flow model for cell motility

    Science.gov (United States)

    Kimpton, L. S.; Whiteley, J. P.; Waters, S. L.; Oliver, J. M.

    2016-04-01

    A wide range of biological processes rely on the ability of cells to move through their environment. Mathematical models have been developed to improve our understanding of how cells achieve motion. Here we develop models that explicitly track the cell's distribution of myosin within a two-phase flow framework. Myosin is a small motor protein which is important for contracting the cell's actin cytoskeleton and enabling cell motion. The two phases represent the actin network and the cytosol in the cell. We start from a fairly general description of myosin kinetics, advection and diffusion in the two-phase flow framework, then identify a number of sub-limits of the model that may be relevant in practice, two of which we investigate further via linear stability analyses and numerical simulations. We demonstrate that myosin-driven contraction of the actin network destabilizes a stationary steady state leading to cell motion, but that rapid diffusion of myosin and rapid unbinding of myosin from the actin network are stabilizing. We use numerical simulation to investigate travelling-wave solutions relevant to a steadily gliding cell and we consider a reduction of the model in which the cell adheres strongly to the substrate on which it is crawling. This work demonstrates that a number of existing models for the effect of myosin on cell motility can be understood as different sub-limits of our two-phase flow model.

  1. Genetic variation of the major histocompatibility complex (MHC class II B gene in the threatened Hume's pheasant, Syrmaticus humiae.

    Directory of Open Access Journals (Sweden)

    Weicai Chen

    Full Text Available Major histocompatibility complex (MHC genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae, which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

  2. The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

    Science.gov (United States)

    Noguchi, Taro Q. P.; Morimatsu, Masatoshi; Iwane, Atsuko H.; Yanagida, Toshio; Uyeda, Taro Q. P.

    2015-01-01

    The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin. PMID:25945499

  3. The role of structural dynamics of actin in class-specific myosin motility.

    Directory of Open Access Journals (Sweden)

    Taro Q P Noguchi

    Full Text Available The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

  4. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

    Science.gov (United States)

    Winkelmann, D A; Bourdieu, L; Kinose, F; Libchaber, A

    1995-04-01

    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

  5. Genetic Variation in Myosin 1H Contributes to Mandibular Prognathism

    Science.gov (United States)

    Tassopoulou-Fishell, Maria; Deeley, Kathleen; Harvey, Erika M.; Sciote, James; Vieira, Alexandre R.

    2013-01-01

    Introduction Several candidate loci have been suggested as influencing mandibular prognathism (1p22.1, 1p22.2, 1p36, 3q26.2, 5p13-p12, 6q25, 11q22.2-q22.3, 12q23, 12q13.13, and 19p13.2). The goal of this study was to replicate these results in a well-characterized homogeneous sample set. Methods Thirty-three single nucleotide polymorphisms spanning all candidate regions were studied in 44 prognathic and 35 Class I subjects from the University of Pittsburgh School of Dental Medicine Dental Registry and DNA Repository. The 44 mandibular prognathism subjects had an average age of 18.4 years, 31 were females and 13 males, and 24 were White, 15 African American, two Hispanic, and three Asian. The 35 Class I subjects had an average age of 17.6 years, 27 were females and 9 males, and 27 were White, six African Americans, one Hispanic, and two Asian. Skeletal mandibular prognathism diagnosis included cephalometric values indicative of Class III such as ANB smaller than two degrees, negative Witts appraisal, and positive A–B plane. Additional mandibular prognathism criteria included negative OJ and visually prognathic (concave) profile as determined by the subject's clinical evaluation. Orthognathic subjects without jaw deformations were used as a comparison group. Mandibular prognathism and orthognathic subjects were matched based on race, sex and age. Genetic markers were tested by polymerase chain reaction using TaqMan chemistry. Chi-square and Fisher exact tests were used to determine overrepresentation of marker allele with alpha of 0.05. Results An association was unveiled between a marker in MYO1H (rs10850110) and the mandibular prognathism phenotype (p=0.03). MYO1H is a Class-I myosin that is in a different protein group than the myosin isoforms of muscle sarcomeres, which are the basis of skeletal muscle fiber typing. Class I myosins are necessary for cell motility, phagocytosis and vesicle transport. Conclusions More strict clinical definitions may increase

  6. Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

    Science.gov (United States)

    Rivard, Maxime; Couture, Charles-André; Miri, Amir K; Laliberté, Mathieu; Bertrand-Grenier, Antony; Mongeau, Luc; Légaré, François

    2013-01-01

    We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

  7. Cooperative folding of muscle myosins: I. Mechanical model

    CERN Document Server

    Caruel, Matthieu; Truskinovsky, Lev

    2013-01-01

    Mechanically induced folding of passive cross-linkers is a fundamental biological phenomenon. A typical example is a conformational change in myosin II responsible for the power-stroke in skeletal muscles. In this paper we present an athermal perspective on such folding by analyzing the simplest purely mechanical prototype: a parallel bundle of bi-stable units attached to a common backbone. We show that in this analytically transparent model, characterized by a rugged energy landscape, the ground states are always highly coherent, single-phase configurations. We argue that such cooperative behavior, ensuring collective conformational change, is due to the dominance of long- range interactions making the system non-additive. The detailed predictions of our model are in agreement with experimentally observed non-equivalence of fast force recovery in skeletal muscles loaded in soft and hard devices. Some features displayed by the model are also recognizable in the behavior of other biological systems with passiv...

  8. Sarcomere lattice geometry influences cooperative myosin binding in muscle.

    Directory of Open Access Journals (Sweden)

    Bertrand C W Tanner

    2007-07-01

    Full Text Available In muscle, force emerges from myosin binding with actin (forming a cross-bridge. This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca(2+ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca(2+ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model, while the other comprises only one thick and one thin filament (two-filament model. Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.

  9. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    Science.gov (United States)

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion.

  10. Identification and characterization of the Bombyx mori myosin II essential light chain and its effect in BmNPV infection

    Directory of Open Access Journals (Sweden)

    L Hao

    2015-02-01

    Full Text Available Myosin, as a type of molecular motor, is mainly involved in muscle contraction. Recently, myosin research has made considerable progress. However, the function of Bombyx mori myosin remains unclear. In this study, we cloned the BmMyosin II essential light chain (BmMyosin II ELC gene from a cDNA library of silkworm, which had an open reading frame (ORF of 444 bp encoding 147 amino acids (about 16 kDa. After analyzing their sequences, BmMyosin II ELC was similar to the ELCs of 27 other Myosin II types, which contained EFh domain that bound Ca2+. In addition, 28 sequences had five motifs, motifs 1 and 3 were relatively conserved. We constructed two vectors with BmMyosin to transfect MGC803 or BmN, monolayer wound healing of cells indicated they can promote cell migration successfully. For three fifth instar silkworms, Bm306, BmNB, BmBC8, we mainly analyzed the change of BmMyosin II ELC from transcription and translation after infecting with nucleopolyhedrovirus (BmNPV. We found that gene expression of resistant strains were higher than susceptible strains at 12 h, while the result of the translation level was opposite that of the transcription level. Through in vitro protein interactions, we found BmMyosin II ELC can interact with BmNPV ubiquitin.

  11. Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus.

    Science.gov (United States)

    Sobczak, Magdalena; Wasik, Anna; Kłopocka, Wanda; Redowicz, Maria Jolanta

    2008-12-01

    Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).

  12. Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees.

    Science.gov (United States)

    Patricio, Karina; Calábria, Luciana Karen; Peixoto, Pablo Marco; Espindola, Foued Salmen; Da Cruz-Landim, Carminda

    2010-10-01

    The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved.

  13. Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

    National Research Council Canada - National Science Library

    Sen, Arnab; Nagy-Zsvér-Vadas, Zsanett; Krahn, Michael P

    2012-01-01

    ... (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical-basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation...

  14. Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

    Science.gov (United States)

    Hong, Feng; Facemyer, Kevin C; Carter, Michael S; Jackson, Del R; Haldeman, Brian D; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P; Cremo, Christine R; Baker, Josh E

    2013-11-26

    During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

  15. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  16. Localization and mobility of synaptic vesicles in Myosin VI mutants of Drosophila.

    Directory of Open Access Journals (Sweden)

    Marta Kisiel

    Full Text Available BACKGROUND: At the Drosophila neuromuscular junction (NMJ, synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila. RESULTS: In Drosophila, Myosin VI is encoded by the gene, jaguar (jar. To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching. CONCLUSION: This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.

  17. Myosin types and fiber types in cardiac muscle. II. Atrial myocardium

    OpenAIRE

    1982-01-01

    Antibodies were produced against myosins isolated from the left atrial myocardium (anti-bAm) and the left ventricular myocardium (anti-bVm) of the bovine heart. Cross-reactive antibodies were removed by cross- absorption. Absorbed anti-bAm and anti-bVm were specific for the myosin heavy chains when tested by enzyme immunoassay combined with SDS gel electrophoresis. Indirect immunofluorescence was used to determine the reactivity of atrial muscle fibers to the two antibodies. Three populations...

  18. Single-Molecule Measurement of the Stiffness of the Rigor Myosin Head

    OpenAIRE

    2007-01-01

    The force-extension curve of single myosin subfragment-1 molecules, interacting in the rigor state with an actin filament, has been investigated at low [ATP] by applying a slow triangle-wave movement to the optical traps holding a bead-actin-bead dumbbell. In combination with a measurement of the overall stiffness of the dumbbell, this allowed characterization of the three extensible elements, the actin-bead links and the myosin. Simultaneously, another method, based on an analysis of bead po...

  19. Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

    Science.gov (United States)

    Lelli, Andrea; Michel, Vincent; Boutet de Monvel, Jacques; Cortese, Matteo; Bosch-Grau, Montserrat; Aghaie, Asadollah; Perfettini, Isabelle; Dupont, Typhaine; Avan, Paul; El-Amraoui, Aziz; Petit, Christine

    2016-01-18

    The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.

  20. Kinetic characterization of the ATPase and actin-activated ATPase activities of Acanthamoeba castellanii myosin-2.

    Science.gov (United States)

    Heissler, Sarah M; Liu, Xiong; Korn, Edward D; Sellers, James R

    2013-09-13

    Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

  1. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    Science.gov (United States)

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

  2. Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

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    Lelli, Andrea; Michel, Vincent; Boutet de Monvel, Jacques; Cortese, Matteo; Bosch-Grau, Montserrat; Aghaie, Asadollah; Perfettini, Isabelle; Dupont, Typhaine; Avan, Paul

    2016-01-01

    The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a−/−Myo3b−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a−/−Myo3b−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a−/−Myo3b−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping. PMID:26754646

  3. Role of plant myosins in motile organelles:Is a direct interaction required?

    Institute of Scientific and Technical Information of China (English)

    Limor Buchnik; Mohamad Abu-Abied; Einat Sadot

    2015-01-01

    Plant organel es are highly motile, with speed values of 3–7 mm/s in cel s of land plants and about 20–60 mm/s in characean algal cel s. This movement is believed to be important for rapid distribution of materials around the cel , for the plant’s ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organel es within plant cel s is based on the actin cytoskeleton and its motor proteins the myosins. Most plants express multiple members of two main classes:myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in al kingdoms. Paradoxical y, while it was found that myosins from class XI regulate most organel e movement, it is not quite clear how or even if these motor proteins attach to the organel es whose movement they regulate.

  4. Myosin II controls cellular branching morphogenesis and migration in three dimensions by minimizing cell-surface curvature.

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    Elliott, Hunter; Fischer, Robert S; Myers, Kenneth A; Desai, Ravi A; Gao, Lin; Chen, Christopher S; Adelstein, Robert S; Waterman, Clare M; Danuser, Gaudenz

    2015-02-01

    In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.

  5. Myosin Va participates in acrosomal formation and nuclear morphogenesis during spermatogenesis of Chinese mitten crab Eriocheir sinensis.

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    Xiao Sun

    Full Text Available BACKGROUND: The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: Western blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV and proacrosomal granule (PG. Subsequently, myosin Va localizes within the proacrosomal vesicle (PV formed by PG and EV fusion and locates in the membrane complex (MC at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT and nucleus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis.

  6. Genome-wide identification, splicing, and expression analysis of the myosin gene family in maize (Zea mays).

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    Wang, Guifeng; Zhong, Mingyu; Wang, Jiajia; Zhang, Jushan; Tang, Yuanping; Wang, Gang; Song, Rentao

    2014-03-01

    The actin-based myosin system is essential for the organization and dynamics of the endomembrane system and transport network in plant cells. Plants harbour two unique myosin groups, class VIII and class XI, and the latter is structurally and functionally analogous to the animal and fungal class V myosin. Little is known about myosins in grass, even though grass includes several agronomically important cereal crops. Here, we identified 14 myosin genes from the genome of maize (Zea mays). The relatively larger sizes of maize myosin genes are due to their much longer introns, which are abundant in transposable elements. Phylogenetic analysis indicated that maize myosin genes could be classified into class VIII and class XI, with three and 11 members, respectively. Apart from subgroup XI-F, the remaining subgroups were duplicated at least in one analysed lineage, and the duplication events occurred more extensively in Arabidopsis than in maize. Only two pairs of maize myosins were generated from segmental duplication. Expression analysis revealed that most maize myosin genes were expressed universally, whereas a few members (XI-1, -6, and -11) showed an anther-specific pattern, and many underwent extensive alternative splicing. We also found a short transcript at the O1 locus, which conceptually encoded a headless myosin that most likely functions at the transcriptional level rather than via a dominant-negative mechanism at the translational level. Together, these data provide significant insights into the evolutionary and functional characterization of maize myosin genes that could transfer to the identification and application of homologous myosins of other grasses.

  7. Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure.

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    Manickam, Manoj; Jalani, Hitesh B; Pillaiyar, Thanigaimalai; Sharma, Niti; Boggu, Pulla Reddy; Venkateswararao, Eeda; Lee, You-Jung; Jeon, Eun-Seok; Jung, Sang-Hun

    2017-07-07

    A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Identification, modeling, and characterization studies of Tetrahymena thermophila myosin FERM domains suggests a conserved core fold but functional differences.

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    Martin, Che L; Singh, Shaneen M

    2015-11-01

    Myosins (MYO) define a superfamily of motor proteins which facilitate movement along cytoskeletal actin filaments in an ATP-dependent manner. To date, over 30 classes of myosin have been defined that vary in their roles and distribution across different taxa. The multidomain tail of myosin is responsible for the observed functional differences in different myosin classes facilitating differential binding to different cargos. One domain found in this region, the FERM domain, is found in several diverse proteins and is involved in many biological functions ranging from cell adhesion and actin-driven cytoskeleton assembly to cell signaling. Recently, new classes of unconventional myosin have been identified in Tetrahymena thermophila. In this study, we have identified, modeled, and characterized eight FERM domains from the unconventional T. thermophila myosins as their complete functional MyTH4-FERM cassettes. Our results reveal notable sequence, structural, and electrostatic differences between T. thermophila and other characterized FERM domains. Specifically, T. thermophila FERM domains contain helical inserts or extensions, which contribute to significant differences in surface electrostatic profiles of T. thermophila myosin FERMs when compared to the conventional FERM domains. Analyses of the modeled domains reveal differences in key functional residues as well as phosphoinositide-binding signatures and affinities. The work presented here broadens the scope of our understanding of myosin classes and their inherent functions, and provides a platform for experimentalists to design rational experimental studies to test the functional roles for T. thermophila myosins.

  9. Novel Simulation Model of Non-Muscle Invasive Bladder Cancer: A Platform for a Virtual Randomized Trial of Conservative Therapy vs. Cystectomy in BCG Refractory Patients.

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    Patel, Sanjay; Dinh, Tuan; Noah-Vanhoucke, Joyce; Rengarajan, Badri; Mayo, Kevin; Clark, Peter E; Kamat, Ashish M; Lee, Cheryl T; Sexton, Wade J; Steinberg, Gary D

    2015-10-26

    Introduction: There have been no randomized controlled trials (RCTs) evaluating the clinical or economic benefit of mitomycin C intravesical therapy vs. radical cystectomy in patients with high-risk non-muscle invasive bladder cancer (NMIBC). We used the Archimedes computational model to simulate RCT comparing radical cystectomy versus intravesical mitomycin C (MMC) therapy to evaluate the clinical and economic outcomes for BCG-refractory NMIBC as well demonstrate the utility of computer based models to simulate a clinical trial. Methods: The Archimedes model was developed to generate a virtual population using the Surveillance Epidemiology and End Results database, other clinical trials, and expert opinions. Patients selected were diagnosed with NMIBC (virtual patients were evaluation. Progression to MIBC in the MMC treatment arm was 30% over the lifetime. Disease specific death at 5 years was 1.6% and 8.7% for the immediate cystectomy and MMC treatment arms respectively; while, overall death was 17.8% and 23.8% at 5 years. Over a 5-year period the average cost of immediate cystectomy was $64,675 vs $68,517 in the MMC arm. Conclusion: Immediate radical cystectomy after BCG failure for NMIBC has improved survival and is more cost-effective when compared to those undergoing MMC. Simulation of clinical trials using computational models similar to the Archimedes model can overcome shortcomings of real-world clinical trials and may prove useful in the face of current medical cost-conscious era.

  10. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

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    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  11. Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin.

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    Katsuyuki Shiroguchi

    2011-04-01

    Full Text Available Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(BT of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery

  12. Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

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    Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

    2016-05-24

    Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

  13. Myosin-II-Mediated Directional Migration of Dictyostelium Cells in Response to Cyclic Stretching of Substratum

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    Iwadate, Yoshiaki; Okimura, Chika; Sato, Katsuya; Nakashima, Yuta; Tsujioka, Masatsune; Minami, Kazuyuki

    2013-01-01

    Living cells are constantly subjected to various mechanical stimulations, such as shear flow, osmotic pressure, and hardness of substratum. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. Cells adhering to substrata must receive and respond to mechanical stimuli from the substrata to decide their shape and/or migrating direction. In response to cyclic stretching of the elastic substratum, intracellular stress fibers in fibroblasts and endothelial, osteosarcoma, and smooth muscle cells are rearranged perpendicular to the stretching direction, and the shape of those cells becomes extended in this new direction. In the case of migrating Dictyostelium cells, cyclic stretching regulates the direction of migration, and not the shape, of the cell. The cells migrate in a direction perpendicular to that of the stretching. However, the molecular mechanisms that induce the directional migration remain unknown. Here, using a microstretching device, we recorded green fluorescent protein (GFP)-myosin-II dynamics in Dictyostelium cells on an elastic substratum under cyclic stretching. Repeated stretching induced myosin II localization equally on both stretching sides in the cells. Although myosin-II-null cells migrated randomly, myosin-II-null cells expressing a variant of myosin II that cannot hydrolyze ATP migrated perpendicular to the stretching. These results indicate that Dictyostelium cells accumulate myosin II at the portion of the cell where a large strain is received and migrate in a direction other than that of the portion where myosin II accumulated. This polarity generation for migration does not require the contraction of actomyosin. PMID:23442953

  14. Molecular dynamics simulation for the reversed power stroke motion of a myosin subfragment-1.

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    Masuda, Tadashi

    2015-06-01

    Myosins are typical molecular motor proteins that convert the chemical energy from the ATP hydrolysis into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results already obtained, Masuda has proposed a hypothesis called the "Driven by Detachment" theory for the working principle of the myosins. This theory insists that the energy used during the power stroke of the myosins does not directly originate from the chemical energy of ATP, but is converted from the elastic energy within the molecule at the joint between the head and neck domains. One method for demonstrating the validity of this theory is a computational simulation using the molecular dynamics (MD) method. The MD software used was GROMACS. The target of the MD simulations was myosin subfragment-1 (S1), for which the initial structure was obtained from the Protein Data Bank entry 1M8Q. The AFM pull code of GROMACS was used to apply an external force of 17 pN at the end of the neck domain in the direction opposite to the power stroke to observe whether the myosin S1 takes the pre-power stroke conformation. The residues assumed to be engaged in the docking with an actin filament were fixed to the space. Starting from exactly the same initial position, 10 simulations were repeated by varying the random seeds for generating the initial velocities of the atoms. After 64ns of calculations, the myosin S1 took the conformation of the pre-power stroke state in which the neck domain was bent around the joint between the head and the neck domains. This result agrees with the prediction expected by the DbD theory, the validity of which may be established by conducting similar simulations for the other steps of the myosin working processes. Copyright © 2015. Published by Elsevier Ireland Ltd.

  15. Actomyosin content of Physarum plasmodia and detection of immunological cross-reactions with myosins from related species

    Science.gov (United States)

    1976-01-01

    The content of myosin in plasmodia of the myxomycete Physarum polycephalum was measured by an immunological technique, quantitative microcomplement (C') fixation. Migrating plasmodia (starved after growth on rolled oats) contained 0.60 +/- 0.08 (SD) mg myosin per g fresh plasmodia. Myosin comprised 0.77% +/- 0.05 (SD) of the total plasmodial protein. When total plasmodial proteins were separated by electrophoresis on SDS-polyacrylamide gels, a large amount of protein appeared in a band comigrating with muscle actin. Densitometry performed after Coomassie blue staining indicated that as much as 15- 25% of the total protein in the plasmodium could be actin. This gives an actin/myosin ratio by weight in the myxomycete plasmodium as high as 19-33, a very "actin-rich" actomyosin compared with rabbit skeletal muscle actomyosin with an actin/myosin ratio of 0.6. Starvation stimulates rapid migration and is correlated with a higher percent of both myosin and actin in the total protein of the plasmodium compared with normally growing cultures. Immunological cross-reaction of myosins from a variety of species was measured by C' fixation using an antiserum produced against purified native myosin from P. polycephalum. Although myxomycete and vertebrate striated muscle myosins have very similar morphological and biochemical properties, and apparently possess similar binding properties to F-actin, only myosins from myxomycetes in the order Physarales, rather closely related to P. polycephalum, gave detectable cross-reactions. This finding suggests that many amino acid sequences in myosin have been variable during evolution. PMID:944188

  16. Roles of an unconventional protein kinase and myosin II in amoeba osmotic shock responses.

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    Betapudi, Venkaiah; Egelhoff, Thomas T

    2009-12-01

    The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. Green fluorescent protein (GFP)-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild-type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa.

  17. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

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    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG

    2006-01-01

    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  18. Myosin VI deafness mutation prevents the initiation of processive runs on actin.

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    Pylypenko, Olena; Song, Lin; Shima, Ai; Yang, Zhaohui; Houdusse, Anne M; Sweeney, H Lee

    2015-03-17

    Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.

  19. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

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    Diana Spitznagel

    Full Text Available Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1 that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  20. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

    Science.gov (United States)

    Spitznagel, Diana; O'Rourke, John F; Leddy, Neal; Hanrahan, Orla; Nolan, Derek P

    2010-01-01

    Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  1. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

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    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F

    1998-04-21

    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

  2. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

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    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  3. An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight.

    Science.gov (United States)

    Wang, Qian; Newhard, Christopher S; Ramanath, Seemanti; Sheppard, Debra; Swank, Douglas M

    2014-01-15

    Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.

  4. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity.

    Science.gov (United States)

    Jana, Biman; Onuchic, José N

    2016-08-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities.

  5. Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

    Science.gov (United States)

    Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

    1992-01-01

    This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

  6. Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

    National Research Council Canada - National Science Library

    Wang, Yang; Melkani, Girish C; Suggs, Jennifer A; Melkani, Anju; Kronert, William A; Cammarato, Anthony; Bernstein, Sanford I

    2012-01-01

    .... We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers...

  7. Criticalities in crosslinked actin networks due to myosin activity

    Science.gov (United States)

    Sheinman, Michael

    2013-03-01

    Many essential processes in cells and tissues, like motility and morphogenesis, are orchestrated by molecular motors applying internal, active stresses on crosslinked networks of actin filaments. Using scaling analysis, mean-field calculation, numerical modelling and in vitro experiments of such active networks we predict and observe different mechanical regimes exhibiting interesting critical behaviours with non-trivial power-law dependencies. Firstly, we find that the presence of active stresses can dramatically increase the stiffness of a floppy network, as was observed in reconstituted intracellular F-actin networks with myosin motors and extracellular gels with contractile cells. Uniform internal stress results in an anomalous, critical mechanical regime only in the vicinity of the rigidity percolation points of the network. However, taking into account heterogeneity of motors, we demonstrate that the motors, stiffening any floppy network, induce large non-affine fluctuations, giving rise to a critical mechanical regime. Secondly, upon increasing motor concentration, the resulting large internal stress is able to significantly enhance unbinding of the network's crosslinks and, therefore, disconnect the initially well-connected network to isolated clusters. However, during this process, when the network approaches marginal connectivity the internal stresses are expected to drop drastically such that the connectivity stabilizes. This general argument and detailed numerical simulations show that motors should drive a well connected network to a close vicinity of a critical point of marginal connectivity. Experiments clearly confirm this conclusion and demonstrate robust critical connectivity of initially well-connected networks, ruptured by the motor activity for a wide range of parameters. M. Sheinman, C.P. Broedersz and F.C. MacKintosh, Phys. Rev. Lett, in press. J. Alvarado, M. Sheinman, A. Sharma, F.C. MacKintosh and G. Koenderink, in preparation.

  8. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    Energy Technology Data Exchange (ETDEWEB)

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  9. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Science.gov (United States)

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  10. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  11. A comparison of intraspecific patterns of DNA sequence variation in mitochondrial DNA, alpha-enolase, and MHC class II B loci in auklets (Charadriiformes: Alcidae).

    Science.gov (United States)

    Walsh, Hollie E; Friesen, Vicki L

    2003-12-01

    Patterns of DNA sequence variation can be used to learn about mechanisms of organismal evolution, but only if mechanisms of sequence evolution are well understood. Although theories of molecular evolution are well developed, few empirical studies have addressed patterns and mechanisms of sequence evolution in nuclear genes within species. In the present study, we compared DNA sequences among three loci with different evolutionary constraints to determine the influences of effective population size, balancing selection, and linkage on intraspecific patterns of sequence variation. Specifically, we assessed the degree and nature of polymorphism in a 307-base pair (bp) fragment of the mitochondrial cytochrome b gene, intron VIII of the gene for alpha-enolase (a presumably neutral nuclear gene), and an approximately 600-bp fragment of an MHC class II B gene, including 155 bp of the hypervariable peptide binding region (a nuclear locus thought to be under balancing selection) for least and crested auklets (Aethia pusilla and A. cristatella; Charadriiformes: Alcidae). Transspecies polymorphism was found in both alpha-enolase and the MHC but not cytochrome b and, given estimates of effective population size, probably represents retained ancestral variation. Biases in nucleotide composition suggested that mutational bias, tRNA availability, and the secondary structure of mRNA and/or DNA may influence base usage. Several lines of evidence indicated that balancing selection may be acting on the MHC II B exon 2. However, no evidence of balancing selection was observed in the intron and exon sequences immediately downstream of MHC II B exon 2.

  12. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  13. Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state.

    Science.gov (United States)

    Burgess, Stan A; Yu, Shuizi; Walker, Matt L; Hawkins, Rhoda J; Chalovich, Joseph M; Knight, Peter J

    2007-10-05

    Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.

  14. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Directory of Open Access Journals (Sweden)

    Valera V Peremyslov

    Full Text Available Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI, cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  15. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A. (UPENN); (Duke); (MRCLMB); (FSU); (Jikei-Med)

    2010-10-22

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are

  16. Aberrant post-translational modifications compromise human myosin motor function in old age.

    Science.gov (United States)

    Li, Meishan; Ogilvie, Hannah; Ochala, Julien; Artemenko, Konstantin; Iwamoto, Hiroyuki; Yagi, Naoto; Bergquist, Jonas; Larsson, Lars

    2015-04-01

    Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

  17. Clinical significance and pathogenic role of anti-cardiac myosin autoantibody in dilated cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective In order to explore the possible roles played by the autoimmune mechanism in the progression of myocarditis into dilated cardiomyopathy (DCM) using an animal model, we investigated whether autoimmune myocarditis might develop into DCM. Methods Experimental Balb/C mice (n=20) were immunized with cardiac myosin with Freund's complete adjuvant at days 0, 7 and 30. The control Balb/C mice (n=10) were immunized with Freund's complete adjuvant in the same mannere. Serum and myocardium samples were collected after the first immunization at days 15, 21 and 120. The anti-myosin antibody was examined by enzyme-linked immunosorbent assay and immunoblotting.Results Pathological findings demonstrated that there was myocardial necrosis or inflammatory infiltration during acute stages and fibrosis mainly in the late phase of experimental group, but the myocardial lesions were not found in the control group. Autoimmunity could induce myocarditis and DCM in the absence of viral infection. High titer anti-myosin IgG antibodies were found in the experimental group, but not in the control group. Furthermore, the anti-myosin heavy chain (200 KD) antibody was positive in 21 of 48 patients with DCM and viral myocarditis, but only 4 of 20 patients with coronary heart disease, including 1 case and 3 cases that reacted with heavy and light chains (27.5 KD), respectively. The antibodies were not detected in healthy donors.Conclusion Cardiac myosin might be an autoantigen that provokes autoimmunity and leads to the transformation of myocarditis into DCM. Detection of anti-myosin heavy chain antibody might contribute to diagnosis for DCM and viral myocarditis.

  18. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Directory of Open Access Journals (Sweden)

    Shenping Wu

    Full Text Available Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  19. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Science.gov (United States)

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  20. Humoral immune response against contractile proteins (actin and myosin) during cardiovascular disease.

    Science.gov (United States)

    De Scheerder, I K; De Buyzere, M; Delanghe, J; Maas, A; Clement, D L; Wieme, R

    1991-08-01

    Sensitive and highly specific ELISA assays were developed to determine humoral immune response against actin and myosin in 122 patients suffering from various cardiovascular diseases: acute viral myocarditis (n = 10, MYO), acute myocardial infarction (n = 28, AMI), valve surgery (n = 35, VALVE), coronary bypass surgery (n = 35, CABG), and peripheral vascular surgery (n = 14, VASC). Anti-actin and anti-myosin antibodies were determined on admission and serially during a period of 90 days. Anti-actin and anti-myosin immune response (IgG, IgM) was expressed comparing absorbance of the patients' serum with a reference serum. In the different patient groups significantly (P less than 0.01) higher anti-actin and anti-myosin antibody concentrations were found on admission compared with age-matched control groups. During follow-up, all patient groups except the vascular surgery group showed a significant immune response against actin and myosin, with an immune response ratio (peak/admission) for AMA IgG and IgM respectively of 2.12 and 2.40 in the VALVE group, 1.30 and 1.99 in the CABG group, 1.42 and 1.48 in the AMI group and 1.66 and 1.25 in the MYO group; and for AAA IgG and IgM respectively of 1.57 and 3.00 in the VALVE group, 1.54 and 1.64 in the CABG group, 1.25 and 1.07 in the AMI group, and 1.42 and 1.42 in the MYO group. A significant correlation between pre-cardiac injury and peak post-cardiac injury anti-myosin and anti-actin autoantibody levels could be demonstrated suggesting that pre-injury sensitization to these antigens plays an important role in evoking post-cardiac injury immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. p53 Status correlates with the risk of recurrence in non-muscle invasive bladder cancers treated with Bacillus Calmette-Guerin: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Xiaofeng Zhou

    Full Text Available Published studies have yielded inconsistent results on the relationship between p53 status and the prognosis of non-muscle invasive bladder cancer (NMIBC treated with Bacillus Calmette-Guérin (BCG intravesical therapy. Therefore, we performed a meta-analysis to evaluate the prognostic value of p53 in NMIBC treated with BCG.We systematically searched for relevant literature in PubMed, EMBASE, CNKI, and Chinese Wanfang databases. Hazard ratios (HRs with 95% confidence intervals (CIs were combined as the effect size (ES across studies for recurrence-free survival (RFS and progression-free survival (PFS.A total of 11 studies, consisting of 1,049 participants, met the criteria. Overall, there was no clear relationship between p53 status and RFS or PFS for NMIBC patients treated with BCG (HR: 1.40, 95% CI: 0.91-2.16; HR: 1.37, 95% CI: 0.90-2.09, respectively. Obvious heterogeneity was observed across the studies (I2 = 69.5%, P = 0.001; I2 = 44.7%, P = 0.081, respectively. In stratified analysis by region, p53 overexpression was a predictor of poor RFS in Asian populations (HR: 1.57, 95% CI: 1.08-2.27. In addition, after excluding the studies that possibly contributed to the heterogeneity by the Galbraith plot, the overall association for RFS became statistically significant (HR: 1.38 95% CI: 1.08-1.77 without evidence of heterogeneity (I2 = 0.0%, P = 0.499.This meta-analysis suggests that p53 overexpression in NMIBC patients treated with BCG may be associated with RFS, especially in Asian populations. Because of the heterogeneity and other limitations, further studies with rigid criteria and large populations are still warranted to confirm our findings.

  2. Summary and Recommendations from the National Cancer Institute's Clinical Trials Planning Meeting on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer.

    Science.gov (United States)

    Lerner, Seth P; Bajorin, Dean F; Dinney, Colin P; Efstathiou, Jason A; Groshen, Susan; Hahn, Noah M; Hansel, Donna; Kwiatkowski, David; O'Donnell, Michael; Rosenberg, Jonathan; Svatek, Robert; Abrams, Jeffrey S; Al-Ahmadie, Hikmat; Apolo, Andrea B; Bellmunt, Joaquim; Callahan, Margaret; Cha, Eugene K; Drake, Charles; Jarow, Jonathan; Kamat, Ashish; Kim, William; Knowles, Margaret; Mann, Bhupinder; Marchionni, Luigi; McConkey, David; McShane, Lisa; Ramirez, Nilsa; Sharabi, Andrew; Sharpe, Arlene H; Solit, David; Tangen, Catherine M; Amiri, Abdul Tawab; Van Allen, Eliezer; West, Pamela J; Witjes, J A; Quale, Diane Zipursky

    2016-04-27

    The NCI Bladder Cancer Task Force convened a Clinical Trials Planning Meeting (CTPM) Workshop focused on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer (NMIBC). Meeting attendees included a broad and multi-disciplinary group of clinical and research stakeholders and included leaders from NCI, FDA, National Clinical Trials Network (NCTN), advocacy and the pharmaceutical and biotech industry. The meeting goals and objectives were to: 1) create a collaborative environment in which the greater bladder research community can pursue future optimally designed novel clinical trials focused on the theme of molecular targeted and immune-based therapies in NMIBC; 2) frame the clinical and translational questions that are of highest priority; and 3) develop two clinical trial designs focusing on immunotherapy and molecular targeted therapy. Despite successful development and implementation of large Phase II and Phase III trials in bladder and upper urinary tract cancers, there are no active and accruing trials in the NMIBC space within the NCTN. Disappointingly, there has been only one new FDA approved drug (Valrubicin) in any bladder cancer disease state since 1998. Although genomic-based data for bladder cancer are increasingly available, translating these discoveries into practice changing treatment is still to come. Recently, major efforts in defining the genomic characteristics of NMIBC have been achieved. Aligned with these data is the growing number of targeted therapy agents approved and/or in development in other organ site cancers and the multiple similarities of bladder cancer with molecular subtypes in these other cancers. Additionally, although bladder cancer is one of the more immunogenic tumors, some tumors have the ability to attenuate or eliminate host immune responses. Two trial concepts emerged from the meeting including a window of opportunity trial (Phase 0) testing an FGFR3 inhibitor and a second multi-arm multi-stage trial testing combinations

  3. A meta-analysis of narrow band imaging for the diagnosis and therapeutic outcome of non-muscle invasive bladder cancer

    Science.gov (United States)

    Ma, ShuJuan; Ge, Jing; Zhou, LiZhi; Li, Dongliang; Chen, Qing

    2017-01-01

    Objectives To assess the additional detection rate (ADR) of within-patient comparisons of Narrow band imaging (NBI) and white light cystoscopy (WLC) for non-muscle invasive bladder cancer (NMIBC) detection and compare the impact of NBI and WLC on bladder cancer recurrence risk. Methods We searched relevant studies from PubMed, Embase, Medline, Web of Science and the Cochrane Library database for all articles in English published beforeJuly26th, 2016. Pooled ADR, diagnostic accuracy, relative risk (RR) and their 95% confidence intervals (CIs) were calculated. Results Twenty-five studies including 17 full texts and eight meeting abstracts were included for analysis. Compared to WLC, pooled ADR of NBI for NMIBC diagnosis was 9.9% (95% CI: 0.05–0.14) and 18.6% (95% CI: 0.15–0.25) in per-patient and per-lesion analysis, respectively. Pooled ADR of NBI for carcinoma in situ (CIS) diagnosis was 25.1% (95% CI: 0.09–0.42) and 31.1% (95% CI: 0.24–0.39) for per-patient and per-lesion analyses, respectively. The pooled sensitivity of NBI was significantly higher than WLC both at the per-patient (95.8% vs. 81.6%) and per-lesion levels (94.8% vs. 72.4%). In addition, NBI significantly reduced the recurrence rate of bladder cancer with a pooled RR value of 0.43 (95% CI: 0.23–0.79) and0.81 (95% CI: 0.69–0.95) at month three and twelve, respectively. Conclusions NBI is a valid technique that improves the diagnosis of NMIBC and CIS compared to standard WLC either at per-patient or per-lesion level. It can reduce the recurrence rate of bladder cancer accordingly. PMID:28192481

  4. An evaluation of morphological and functional multi-parametric MRI sequences in classifying non-muscle and muscle invasive bladder cancer.

    Science.gov (United States)

    Panebianco, Valeria; De Berardinis, Ettore; Barchetti, Giovanni; Simone, Giuseppe; Leonardo, Constantino; Grompone, Marcello Domenico; Del Monte, Maurizio; Carano, Davide; Gallucci, Michele; Catto, James; Catalano, Carlo

    2017-09-01

    Our goal is to determine the ability of multi-parametric magnetic resonance imaging (mpMRI) to differentiate muscle invasive bladder cancer (MIBC) from non-muscle invasive bladder cancer (NMIBC). Patients underwent mpMRI before tumour resection. Four MRI sets, i.e. T2-weighted (T2W) + perfusion-weighted imaging (PWI), T2W plus diffusion-weighted imaging (DWI), T2W + DWI + PWI, and T2W + DWI + PWI + dif-fusion tensor imaging (DTI) were interpreted qualitatively by two radiologists, blinded to histology results. PWI, DWI and DTI were also analysed quantitatively. Accuracy was determined using histopathology as the reference standard. A total of 82 tumours were analysed. Ninety-six percent of T1-labeled tumours by the T2W + DWI + PWI image set were confirmed to be NMIBC at histopathology. Overall accuracy of the complete mpMRI protocol was 94% in differentiating NMIBC from MIBC. PWI, DWI and DTI quantitative parameters were shown to be significantly different in cancerous versus non-cancerous areas within the bladder wall in T2-labelled lesions. MpMRI with DWI and DTI appears a reliable staging tool for bladder cancer. If our data are validated, then mpMRI could precede cystoscopic resection to allow a faster recognition of MIBC and accelerated treatment pathways. • A critical step in BCa staging is to differentiate NMIBC from MIBC. • Morphological and functional sequences are reliable techniques in differentiating NMIBC from MIBC. • Diffusion tensor imaging could be an additional tool in BCa staging.

  5. Summary and Recommendations from the National Cancer Institute’s Clinical Trials Planning Meeting on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer

    Science.gov (United States)

    Lerner, Seth P.; Bajorin, Dean F.; Dinney, Colin P.; Efstathiou, Jason A.; Groshen, Susan; Hahn, Noah M.; Hansel, Donna; Kwiatkowski, David; O’Donnell, Michael; Rosenberg, Jonathan; Svatek, Robert; Abrams, Jeffrey S.; Al-Ahmadie, Hikmat; Apolo, Andrea B.; Bellmunt, Joaquim; Callahan, Margaret; Cha, Eugene K.; Drake, Charles; Jarow, Jonathan; Kamat, Ashish; Kim, William; Knowles, Margaret; Mann, Bhupinder; Marchionni, Luigi; McConkey, David; McShane, Lisa; Ramirez, Nilsa; Sharabi, Andrew; Sharpe, Arlene H.; Solit, David; Tangen, Catherine M.; Amiri, Abdul Tawab; Van Allen, Eliezer; West, Pamela J.; Witjes, J. A.; Quale, Diane Zipursky

    2016-01-01

    The NCI Bladder Cancer Task Force convened a Clinical Trials Planning Meeting (CTPM) Workshop focused on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer (NMIBC). Meeting attendees included a broad and multi-disciplinary group of clinical and research stakeholders and included leaders from NCI, FDA, National Clinical Trials Network (NCTN), advocacy and the pharmaceutical and biotech industry. The meeting goals and objectives were to: 1) create a collaborative environment in which the greater bladder research community can pursue future optimally designed novel clinical trials focused on the theme of molecular targeted and immune-based therapies in NMIBC; 2) frame the clinical and translational questions that are of highest priority; and 3) develop two clinical trial designs focusing on immunotherapy and molecular targeted therapy. Despite successful development and implementation of large Phase II and Phase III trials in bladder and upper urinary tract cancers, there are no active and accruing trials in the NMIBC space within the NCTN. Disappointingly, there has been only one new FDA approved drug (Valrubicin) in any bladder cancer disease state since 1998. Although genomic-based data for bladder cancer are increasingly available, translating these discoveries into practice changing treatment is still to come. Recently, major efforts in defining the genomic characteristics of NMIBC have been achieved. Aligned with these data is the growing number of targeted therapy agents approved and/or in development in other organ site cancers and the multiple similarities of bladder cancer with molecular subtypes in these other cancers. Additionally, although bladder cancer is one of the more immunogenic tumors, some tumors have the ability to attenuate or eliminate host immune responses. Two trial concepts emerged from the meeting including a window of opportunity trial (Phase 0) testing an FGFR3 inhibitor and a second multi-arm multi-stage trial testing combinations

  6. Origin and central projections of rat dorsal penile nerve: possible direct projection to autonomic and somatic neurons by primary afferents of nonmuscle origin.

    Science.gov (United States)

    Núñez, R; Gross, G H; Sachs, B D

    1986-05-22

    Cell number, size, and somatotopic arrangement within the spinal ganglia of the cells of origin of the rat dorsal penile nerve (DPN), and their spinal cord projections, were studied by loading the proximal stump of the severed DPN with horseradish peroxidase (HRP). The DPN sensory cells were located entirely in the sixth lumbar (L6) dorsal root ganglia (DRG), in which a mean of 468 +/- 78 cells per side were observed, measuring 26.7 +/- 0.8 microns in their longest axis (range 10-65 microns) and distributed apparently randomly within the ganglia. Within the spinal cord, no retrograde label was found, i.e., no motoneurons were labeled, indicating that in the rat the DPN is formed exclusively of sensory nerve fibers. Although labeled fibers entered the cord only through L6, transganglionically transported HRP was evident in all spinal segments examined, i.e., T13-S2. Labeled fibers projected along the inner edge of the dorsal horn (medial pathway) throughout their extensive craniosacral distribution. However, laminar distribution varied with spinal segment. In the dorsal horn, terminals or preterminal axons were found in the dorsal horn marginal zone (lamina I), the substantia gelatinosa (lamina II), the nucleus proprius (laminae III and IV--the most consistent projection), Clarke's column (lamina VI), and the dorsal gray commissure. In the ventral horn, terminals were found in lamina VII and lamina IX. Label apposed to cell somas and dendrites in lamina VII may represent direct primary afferent projections onto sympathetic autonomic neurons. In lamina IX, labeled terminals delineated the somas and dendrites of cells that appeared to be motoneurons. This is the first description of an apparently monosynaptic contact onto motoneurons by a primary afferent of nonmuscle origin.

  7. Side population in human non-muscle invasive bladder cancer enriches for cancer stem cells that are maintained by MAPK signalling.

    Directory of Open Access Journals (Sweden)

    Anastasia C Hepburn

    Full Text Available Side population (SP and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC, multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148, and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2(hi relative to the non-SP (NSP fraction (ABCG2(low. ABCG2(hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2 and a three-fold increase in colony forming efficiency (CFE in comparison to ABCG2(low NSP cells. In vivo, ABCG2(hi SP cells enriched for tumour growth compared with ABCG2(low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2(hi SP cells and MEK inhibition also inhibited the ABCG2(hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2(hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.

  8. Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype.

    Science.gov (United States)

    Schubert, Timm; Gleiser, Corinna; Heiduschka, Peter; Franz, Christoph; Nagel-Wolfrum, Kerstin; Sahaboglu, Ayse; Weisschuh, Nicole; Eske, Gordon; Rohbock, Karin; Rieger, Norman; Paquet-Durand, François; Wissinger, Bernd; Wolfrum, Uwe; Hirt, Bernhard; Singer, Wibke; Rüttiger, Lukas; Zimmermann, Ulrike; Knipper, Marlies

    2015-10-01

    The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms.

  9. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

    OpenAIRE

    1999-01-01

    To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles expre...

  10. Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite.

    Science.gov (United States)

    Tiago, Teresa; Simão, Sónia; Aureliano, Manuel; Martín-Romero, Francisco Javier; Gutiérrez-Merino, Carlos

    2006-03-21

    Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys(707) labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP.V(1) to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

  11. Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1.

    Directory of Open Access Journals (Sweden)

    Daria S Logvinova

    Full Text Available Myosin head (myosin subfragment 1, S1 consists of two major structural domains, the motor (or catalytic domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1, we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform, which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.

  12. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS.

  13. Visualizing Key Hinges and a Potential Major Source of Compliance in the Lever Arm of Myosin

    Energy Technology Data Exchange (ETDEWEB)

    J Brown; V Senthil Kumar; E ONeall-Hennessey; L Reshetnikova; H Robinson; M Nguyen-McCarty; A Szent-Gyorgyi; C Cohen

    2011-12-31

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  14. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    KAUST Repository

    Conti, Antonio

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  15. Myosin types and fiber types in cardiac muscle. II. Atrial myocardium.

    Science.gov (United States)

    Gorza, L; Sartore, S; Schiaffino, S

    1982-12-01

    Antibodies were produced against myosins isolated from the left atrial myocardium (anti-bAm) and the left ventricular myocardium (anti-bVm) of the bovine heart. Cross-reactive antibodies were removed by cross-absorption. Absorbed anti-bAm and anti-bVm were specific for the myosin heavy chains when tested by enzyme immunoassay combined with SDS gel electrophoresis. Indirect immunofluorescence was used to determine the reactivity of atrial muscle fibers to the two antibodies. Three populations of atrial muscle fibers were distinguished in the bovine heart: (a) fibers reactive with anti-bAm and unreactive with anti-bVm, like most fibers in the left atrium; (b) fibers reactive with both antibodies, especially numerous in the right atrium; (c) fibers reactive with anti-bVm and unreactive with anti-bAm, present only in the interatrial septum and in specific regions of the right atrium, such as the crista terminalis. These findings can be accounted for by postulating the existence of two distinct types of atrial myosin heavy chains, one of which is antigenically related to ventricular myosin. The tendency for fibers labeled by anti-bVm to occur frequently in bundles and their preferential distribution in the crista terminalis, namely along one of the main conduction pathways between the sinus node and the atrioventricular node, and in the interatrial septum, where different internodal tracts are known to converge, suggests that these fibers may be specialized for faster conduction.

  16. Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Kirk L Mecklenburg

    Full Text Available In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP. Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A. In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

  17. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    Science.gov (United States)

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  18. An embryonic myosin isoform enables stretch activation and cyclical power in Drosophila jump muscle.

    Science.gov (United States)

    Zhao, Cuiping; Swank, Douglas M

    2013-06-18

    The mechanism behind stretch activation (SA), a mechanical property that increases muscle force and oscillatory power generation, is not known. We used Drosophila transgenic techniques and our new muscle preparation, the jump muscle, to determine if myosin heavy chain isoforms influence the magnitude and rate of SA force generation. We found that Drosophila jump muscles show very low SA force and cannot produce positive power under oscillatory conditions at pCa 5.0. However, we transformed the jump muscle to be moderately stretch-activatable by replacing its myosin isoform with an embryonic isoform (EMB). Expressing EMB, jump muscle SA force increased by 163% and it generated net positive power. The rate of SA force development decreased by 58% with EMB expression. Power generation is Pi dependent as >4 mM Pi was required for positive power from EMB. Pi increased EMB SA force, but not wild-type SA force. Our data suggest that when muscle expressing EMB is stretched, EMB is more easily driven backward to a weakly bound state than wild-type jump muscle. This increases the number of myosin heads available to rapidly bind to actin and contribute to SA force generation. We conclude that myosin heavy chain isoforms influence both SA kinetics and SA force, which can determine if a muscle is capable of generating oscillatory power at a fixed calcium concentration. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Myosin heavy chain expression can vary over the length of jaw and leg muscles

    NARCIS (Netherlands)

    Korfage, J.A.M.; Kwee, K.E.; Everts, V.; Langenbach, G.E.J.

    2016-01-01

    Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the

  20. Study on the Tripolyphosphatase (TPPase) Property of Bighead Carp (Aristichthys nobilis) Myosin Subfragment-1

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Myosin subfragment-1 was prepared from the myofibrils of bighead carp (Aristichthys nobilis). The myosin subfragment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30C and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2+ was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2+ under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3molL-1 under the condition of 17mmolL-1 Mg2+. The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a KM of 3.2 mmolL-1.

  1. Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

    Science.gov (United States)

    Mueller, Matthias P; Goody, Roger S

    2016-08-01

    Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016.

  2. Myosin II does not contribute to wound repair in Dictyostelium cells

    Directory of Open Access Journals (Sweden)

    Shigehiko Yumura

    2014-09-01

    Full Text Available Cells are always subjected to mechanical stresses, resulting in wounds of the cell membrane, but cells are able to repair and reseal their wounded membrane. Previous reports have shown that actin and myosin II accumulate around the wound and that the constriction of this purse-string closes the membrane pore. Here, we developed a microsurgical wound assay to assess wound repair in Dictyostelium cells. Fluorescent dye that had been incorporated into the cells leaked out for only 2–3 sec after wounding, and a GFP-derived, fluorescent Ca2+ sensor showed that intracellular Ca2+ transiently increased immediately after wounding. In the absence of external Ca2+, the cell failed to repair itself. During the repair process, actin accumulated at the wounded sites but myosin II did not. The wounds were repaired even in myosin II null cells to a comparable degree as the wild-type cells, suggesting that myosin II does not contribute to wound repair. Thus, the actomyosin purse-string constriction model is not a common mechanism for wound repair in eukaryotic cells, and this discrepancy may arise from the difference in cell size.

  3. Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activity.

    Science.gov (United States)

    Tiago, Teresa; Martel, Paulo; Gutiérrez-Merino, Carlos; Aureliano, Manuel

    2007-04-01

    Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V(10)O(28)(6-)) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V(10) binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V(10) oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V(10) to bind at the back-door, but only on the "open" structures where there is access to the phosphate binding-loop. It is suggested that V(10) acts as a "back-door stop" blocking the closure of the 50-kDa cleft necessary to carry out ATP-gamma-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V(10) and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.

  4. Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, J.H.; Robinson, H.; Senthil Kumar, V. S.; O' Neall-Hennessey, E.; Reshetnikova, L.; Nguyen-McCarty, M.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-01-04

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  5. Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

    This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

  6. Solubilisation of myosin in a solution of low ionic strength L-histidine: Significance of the imidazole ring.

    Science.gov (United States)

    Chen, Xing; Zou, Yufeng; Han, Minyi; Pan, Lihua; Xing, Tong; Xu, Xinglian; Zhou, Guanghong

    2016-04-01

    Myosin, a major muscle protein, can be solubilised in a low ionic strength solution containing L-histidine (His). To elucidate which chemical constituents in His are responsible for this solubilisation, we investigated the effects of 5mM His, imidazole (Imi), L-α-alanine (Ala), 1-methyl-L-histidine (M-his) and L-carnosine (Car) on particle properties of myosin suspensions and conformational characteristics of soluble myosin at low ionic strength (1 mM KCl, pH 7.5). His, Imi and Car, each containing an imidazole ring, were able to induce a myosin suspension, which had small particle size species and high absolute zeta potential, thus increasing the solubility of myosin. His, Imi and Car affected the tertiary structure and decreased the α-helix content of soluble myosin. Therefore, the imidazole ring of His appeared to be the significant chemical constituent in solubilising myosin at low ionic strength solution, presumably by affecting its secondary structure.

  7. X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns

    Science.gov (United States)

    Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto; Suggs, Jennifer A.; Bernstein, Sanford I.

    2014-01-01

    Fruit fly (Drosophila melanogaster) is one of the most useful animal models to study the causes and effects of hereditary diseases because of its rich genetic resources. It is especially suitable for studying myopathies caused by myosin mutations, because specific mutations can be induced to the flight muscle-specific myosin isoform, while leaving other isoforms intact. Here we describe an X-ray-diffraction-based method to evaluate the structural effects of mutations in contractile proteins in Drosophila indirect flight muscle. Specifically, we describe the effect of the headless myosin mutation, Mhc10-Y97, in which the motor domain of the myosin head is deleted, on the X-ray diffraction pattern. The loss of general integrity of the filament lattice is evident from the pattern. A striking observation, however, is the prominent meridional reflection at d = 14.5 nm, a hallmark for the regularity of the myosin-containing thick filament. This reflection has long been considered to arise mainly from the myosin head, but taking the 6th actin layer line reflection as an internal control, the 14.5-nm reflection is even stronger than that of wild-type muscle. We confirmed these results via electron microscopy, wherein image analysis revealed structures with a similar periodicity. These observations have major implications on the interpretation of myosin-based reflections. PMID:25400584

  8. Motor-motor interactions in ensembles of muscle myosin: using theory to connect single molecule to ensemble measurements

    Science.gov (United States)

    Walcott, Sam

    2013-03-01

    Interactions between the proteins actin and myosin drive muscle contraction. Properties of a single myosin interacting with an actin filament are largely known, but a trillion myosins work together in muscle. We are interested in how single-molecule properties relate to ensemble function. Myosin's reaction rates depend on force, so ensemble models keep track of both molecular state and force on each molecule. These models make subtle predictions, e.g. that myosin, when part of an ensemble, moves actin faster than when isolated. This acceleration arises because forces between molecules speed reaction kinetics. Experiments support this prediction and allow parameter estimates. A model based on this analysis describes experiments from single molecule to ensemble. In vivo, actin is regulated by proteins that, when present, cause the binding of one myosin to speed the binding of its neighbors; binding becomes cooperative. Although such interactions preclude the mean field approximation, a set of linear ODEs describes these ensembles under simplified experimental conditions. In these experiments cooperativity is strong, with the binding of one molecule affecting ten neighbors on either side. We progress toward a description of myosin ensembles under physiological conditions.

  9. Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis.

    Directory of Open Access Journals (Sweden)

    Daishi Kitazawa

    Full Text Available Peripheral microtubules (MTs near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

  10. Size and speed of the working stroke of cardiac myosin in situ.

    Science.gov (United States)

    Caremani, Marco; Pinzauti, Francesca; Reconditi, Massimo; Piazzesi, Gabriella; Stienen, Ger J M; Lombardi, Vincenzo; Linari, Marco

    2016-03-29

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.

  11. Actin and nuclear myosin Ⅰ are associated with RNAP Ⅱ and function in gene transcription

    Institute of Scientific and Technical Information of China (English)

    ZHU XiaoJuan; HUANG BaiQu; WANG XingZhi; HAO Shui; ZENG XianLu

    2007-01-01

    The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF,which are required for maximal ATPase activity of the Brg1 component of the BAF complex. Moreover,the essential roles of acfin in transcription mediated by RNA polymerases Ⅰ, Ⅱ and Ⅲ have been demonstrated recently. On the other hand, a myosin Ⅰ isoform, which contains a unique NH2-terminal extension for nucleus localization, has been specifically localized in nucleus. As is well known, myosin Ⅰis an actin-binding protein and plays an important role in various cellular activities. Though actin and nuclear myosin Ⅰ (NM Ⅰ) have been implicated to play distinct roles in gene expression, there has been no evidence for the actin-myosin interaction that might be involved in gene transcription mediated by RNA polymerase Ⅱ (RNAP Ⅱ). Here we show evidence that both actin and NM Ⅰ are associated with RNAP Ⅱ in nucleus by using co-localization and co-IP assays, and they may act together on gene transcription.The antibodies against β-actin or NM Ⅰ can block RNA synthesis in a eukaryotic in vitro transcription system with template DNA comprising the promoter and the coding region of human autocrine motility factor receptor (hAMFR) gene; the antibodies pre-adsorbed with purified actin and NM Ⅰ have no effect in transcriptional inhibition, indicating that the inhibition of transcription by anti-actin and anti-NM Ⅰ is specific. These results suggest a direct involvement of actin-myosin complexes in regulating transcription. It also implicates that actin and NM Ⅰ may co-exist in a same complex with RNAP Ⅱ and the interaction of RNAP Ⅱ with actin and NM Ⅰ functions in the RNAP Ⅱ-mediated transcription.

  12. Modulating myosin restores muscle function in a mouse model of nemaline myopathy.

    Science.gov (United States)

    Lindqvist, Johan; Levy, Yotam; Pati-Alam, Alisha; Hardeman, Edna C; Gregorevic, Paul; Ochala, Julien

    2016-02-17

    Nemaline myopathy, one of the most common congenital myopathies is associated with mutations in various genes including ACTA1. This disease is also characterised by various forms/degrees of muscle weakness with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force-generating capacity of muscle fibres. ACTA1 mutations are also associated with variations in myofibre size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (i) would directly be due to the inefficient actomyosin complex, and (ii) could be restored by manipulating myosin expression. We used a knock-in mouse model expressing the ACTA1 His40Tyr actin mutation found in human patients. We then performed in vivo intramuscular injections of recombinant adeno-associated viral vectors harbouring a myosin transgene known to facilitate muscle contraction. We observed that in presence of the transgene, the intrinsic force-generating capacity was restored and myofibre size was normal. This demonstrates a direct link between disrupted attachment of myosin molecules to actin monomers and muscle fibre atrophy. These data also suggest that further therapeutic interventions should primarily target myosin dysfunction to alleviate the pathology of ACTA1-related nemaline myopathy. This article is protected by copyright. All rights reserved. © 2016 American Neurological Association.

  13. Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic.

    Science.gov (United States)

    Provance, D William; Gourley, Christopher R; Silan, Colleen M; Cameron, L C; Shokat, Kevan M; Goldenring, James R; Shah, Kavita; Gillespie, Peter G; Mercer, John A

    2004-02-17

    Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N(6)-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments.

  14. Determining the impact of oxidation on the motility of single muscle-fibres expressing different myosin isoforms

    DEFF Research Database (Denmark)

    Spanos, Dimitrios; Li, M.; Baron, Caroline P.

    2013-01-01

    Under oxidative stress, myosin has been shown to be one of the muscle proteins that are extensively modified, leading to carbonylation and cross-linking. However, how oxidation affects the actomyosin interaction in muscle fibres with different metabolic profiles and expressing different myosin...... heavy chain (MyHC) isoforms has not been previously investigated. Oxidation of myosin isolated from muscle fibres originating from various porcine muscles with a different metabolic profile was studied using a single muscle fibre in-vitro motility assay, allowing measurements of catalytic properties...

  15. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  16. Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic

    OpenAIRE

    Provance, D. William; Gourley, Christopher R.; Silan, Colleen M.; Cameron, L. C.; Kevan M Shokat; Goldenring, James R.; Shah, Kavita; Gillespie, Peter G.; John A. Mercer

    2004-01-01

    Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N6-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membr...

  17. Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms.

    Science.gov (United States)

    Shaffer, Justin F; Kier, William M

    2016-03-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.

  18. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  19. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    Energy Technology Data Exchange (ETDEWEB)

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-05-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with (..gamma..-/sup 32/P)ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo.

  20. Calix[4]arene C-99 inhibits myosin ATPase activity and changes the organization of contractile filaments of myometrium

    Directory of Open Access Journals (Sweden)

    R. D. Labyntseva,

    2015-12-01

    Full Text Available Calix[4]arenes are cup-like macrocyclic (polyphenolic compounds, they are regarded as promising molecular “platforms” for the design of new physiologically active compounds. We have earlier found that сalix[4]arenе C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus іn vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

  1. Myosin VI Must Dimerize and Deploy Its Unusual Lever Arm in Order to Perform Its Cellular Roles

    Directory of Open Access Journals (Sweden)

    Monalisa Mukherjea

    2014-09-01

    Full Text Available It is unclear whether the reverse-direction myosin (myosin VI functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions.

  2. Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

    Science.gov (United States)

    Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

    2016-01-01

    Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

  3. Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles.

    Science.gov (United States)

    Chen, Yun; Guzik, Stephanie; Sumner, James P; Moreland, John; Koretsky, Alan P

    2011-02-11

    The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.

  4. Myosin-II dependent cell contractility contributes to spontaneous nodule formation of mesothelioma cells

    CERN Document Server

    Tárnoki-Zách, Julia; Méhes, Elod; Paku, Sándor; Neufeld, Zoltán; Hegedus, Balázs; Döme, Balázs; Czirok, Andras

    2015-01-01

    We demonstrate that characteristic nodules emerge in cultures of several malignant pleural mesothelioma (MPM) cell lines. Instead of excessive local cell proliferation, the nodules arise by Myosin II-driven cell contractility. The aggregation process can be prevented or reversed by suitable pharmacological inhibitors of acto-myosin contractility. A cell-resolved elasto-plastic model of the multicellular patterning process indicates that the morphology and size of the nodules as well as the speed of their formation is determined by the mechanical tension cells exert on their neighbors, and the stability of cell-substrate adhesion complexes. A linear stability analysis of a homogenous, self-tensioned Maxwell fluid indicates the unconditional presence of a patterning instability.

  5. A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads

    Science.gov (United States)

    Iwaki, M.; Wickham, S. F.; Ikezaki, K.; Yanagida, T.; Shih, W. M.

    2016-01-01

    Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes. PMID:27941751

  6. Importance of salt and temperature in myosin polymerization during surimi gelation.

    Science.gov (United States)

    Núñez-Flores, Ruth; Cando, Deysi; Borderías, A Javier; Moreno, Helena M

    2018-01-15

    To address the effect of absence of NaCl on myosin heavy chain polymerization during two-step surimi gelation (different setting temperatures/times -5°C/24h and 30°C/30min-followed by heating at 90°C/30min) were considered. In gel samples made without salt (Lot A), no myosin heavy chain (MHC) polymerization was observed, only aggregation, as indicated by the electrophoresis in polyacrylamide/agarose gel profile. Moreover, these gels were characterized by weakly stabilized protein networks as denoted by the dynamic oscillatory measurement and FTIR analysis, resulting in poor quality gels. On the other hand, in gels made with added salt, MHC polymerization occurred, as evidenced by the electrophoresis, and the gelation resulted in a well-stabilized protein network with good physicochemical properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Myosin Specific-T Lymphocytes Mediated Myocardial Inflammation in Adoptive Transferred Rats

    Institute of Scientific and Technical Information of China (English)

    Jin Zhang; Yuhua Liao; Xiang Cheng; Jing Chen; Peng Chen; Xiang Gao; Zhengjenny Zhang

    2006-01-01

    Myosin specific-T lymphocytes might mediate myocardial inflammation and remodeling after AMI. Myosinactivated or unactivated T lymphocytes in vitro were transferred into naǐve syngeneic rats, respectively. T lymphocyte infiltration and myocyte apoptosis were explored by the H&E and TUNNEL. Proteins and mRNA levels of cytokines (IL-1β, IL-6 and TNF-α) in myocardium were determined by RT-PCR and immunohistochemistry. T lymphocyte infiltration was evidently observed after one week of activated T cell transfer. The expressions of cytokines were elevated markedly one week later. The myocyte apoptosis occurred after T lymphocyte infiltration in myocardium. Our findings suggest that cardiac myosin activated-T lymphocytes may mediate myocardial inflammation and remodeling.

  8. A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

    Science.gov (United States)

    Iwaki, M; Wickham, S F; Ikezaki, K; Yanagida, T; Shih, W M

    2016-12-12

    Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

  9. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    persistent ROCK II and guanine triphosphate-bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II-depleted cells but not those lacking ROCK I. These effects originated in part from......The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  10. The Arabidopsis class VIII myosin ATM2 is involved in endocytosis.

    Science.gov (United States)

    Sattarzadeh, Amirali; Franzen, Rainer; Schmelzer, Elmon

    2008-06-01

    Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Copyright 2008 Wiley-Liss, Inc.

  11. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  12. Novel Mutation in the α-Myosin Heavy Chain Gene Is Associated With Sick Sinus Syndrome

    OpenAIRE

    Ishikawa, Taisuke; Jou, Chuanchau J.; Nogami, Akihiko; Kowase, Shinya; Arrington, Cammon B.; Barnett, Spencer M.; Harrell, Daniel T.; Arimura, Takuro; Tsuji, Yukiomi; Kimura, Akinori; Makita, Naomasa

    2015-01-01

    Recent genome-wide association studies have demonstrated an association between MYH6, the gene encoding α-myosin heavy chain (α-MHC), and sinus node function in the general population. Moreover, a rare MYH6 variant, R721W, predisposing susceptibility to sick sinus syndrome has been identified. However, the existence of disease-causing MYH6 mutations for familial sick sinus syndrome and their underlying mechanisms remain unknown. Methods and Results-We screened 9 genotype-negative probands wit...

  13. The compact conformation of the Plasmodium knowlesi myosin tail interacting protein MTIP in complex with the C-terminal helix of myosin A.

    Science.gov (United States)

    Turley, Stewart; Khamrui, Susmita; Bergman, Lawrence W; Hol, Wim G J

    2013-08-01

    The myosin motor of the malaria parasite's invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a "compact" Plasmodium falciparum MTIP•MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an "extended" Plasmodium knowlesi MTIP•MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIP•MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    Science.gov (United States)

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-08-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  15. Smooth muscle myosin inhibition: a novel therapeutic approach for pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    David Ho

    Full Text Available OBJECTIVE: Pulmonary hypertension remains a major clinical problem despite current therapies. In this study, we examine for the first time a novel pharmacological target, smooth muscle myosin, and determine if the smooth muscle myosin inhibitor, CK-2019165 (CK-165 ameliorates pulmonary hypertension. MATERIALS AND METHODS: Six domestic female pigs were surgically instrumented to measure pulmonary blood flow and systemic and pulmonary vascular dynamics. Pulmonary hypertension was induced by hypoxia, or infusion of the thromboxane analog (U-46619, 0.1 µg/kg/min, i.v.. In rats, chronic pulmonary hypertension was induced by monocrotaline. RESULTS: CK-165 (4 mg/kg, i.v. reduced pulmonary vascular resistance by 22±3 and 28±6% from baseline in hypoxia and thromboxane pig models, respectively (p<0.01 and 0.01, while mean arterial pressure also fell and heart rate rose slightly. When CK-165 was delivered via inhalation in the hypoxia model, pulmonary vascular resistance fell by 17±6% (p<0.05 while mean arterial pressure and heart rate were unchanged. In the monocrotaline model of chronic pulmonary hypertension, inhaled CK-165 resulted in a similar (18.0±3.8% reduction in right ventricular systolic pressure as compared with sildenafil (20.3±4.5%. CONCLUSION: Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension.

  16. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

    Directory of Open Access Journals (Sweden)

    Elisama Azevedo

    2012-01-01

    Full Text Available Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

  17. Lack of replication for the myosin-18B association with mathematical ability in independent cohorts.

    Science.gov (United States)

    Pettigrew, K A; Fajutrao Valles, S F; Moll, K; Northstone, K; Ring, S; Pennell, C; Wang, C; Leavett, R; Hayiou-Thomas, M E; Thompson, P; Simpson, N H; Fisher, S E; Whitehouse, A J O; Snowling, M J; Newbury, D F; Paracchini, S

    2015-04-01

    Twin studies indicate that dyscalculia (or mathematical disability) is caused partly by a genetic component, which is yet to be understood at the molecular level. Recently, a coding variant (rs133885) in the myosin-18B gene was shown to be associated with mathematical abilities with a specific effect among children with dyslexia. This association represents one of the most significant genetic associations reported to date for mathematical abilities and the only one reaching genome-wide statistical significance. We conducted a replication study in different cohorts to assess the effect of rs133885 maths-related measures. The study was conducted primarily using the Avon Longitudinal Study of Parents and Children (ALSPAC), (N = 3819). We tested additional cohorts including the York Cohort, the Specific Language Impairment Consortium (SLIC) cohort and the Raine Cohort, and stratified them for a definition of dyslexia whenever possible. We did not observe any associations between rs133885 in myosin-18B and mathematical abilities among individuals with dyslexia or in the general population. Our results suggest that the myosin-18B variant is unlikely to be a main factor contributing to mathematical abilities.

  18. Myosin filament sliding through the Z-disc relates striated muscle fibre structure to function.

    Science.gov (United States)

    Rode, Christian; Siebert, Tobias; Tomalka, Andre; Blickhan, Reinhard

    2016-03-16

    Striated muscle contraction requires intricate interactions of microstructures. The classic textbook assumption that myosin filaments are compressed at the meshed Z-disc during striated muscle fibre contraction conflicts with experimental evidence. For example, myosin filaments are too stiff to be compressed sufficiently by the muscular force, and, unlike compressed springs, the muscle fibres do not restore their resting length after contractions to short lengths. Further, the dependence of a fibre's maximum contraction velocity on sarcomere length is unexplained to date. In this paper, we present a structurally consistent model of sarcomere contraction that reconciles these findings with the well-accepted sliding filament and crossbridge theories. The few required model parameters are taken from the literature or obtained from reasoning based on structural arguments. In our model, the transition from hexagonal to tetragonal actin filament arrangement near the Z-disc together with a thoughtful titin arrangement enables myosin filament sliding through the Z-disc. This sliding leads to swivelled crossbridges in the adjacent half-sarcomere that dampen contraction. With no fitting of parameters required, the model predicts straightforwardly the fibre's entire force-length behaviour and the dependence of the maximum contraction velocity on sarcomere length. Our model enables a structurally and functionally consistent view of the contractile machinery of the striated fibre with possible implications for muscle diseases and evolution.

  19. Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

    Science.gov (United States)

    Mozdziak, P. E.; Greaser, M. L.; Schultz, E.

    1998-01-01

    The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

  20. Myosin-like protein (M(r) 175,000) in Gregarina blaberae.

    Science.gov (United States)

    Ghazali, M; Schrével, J

    1993-01-01

    A myosin-like protein (M(r) 175,000) was detected in the parasitic protozoan Gregarina blaberae, by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. The protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCl) and in the presence of reducing agents (20 mM DTT). The protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the so-called septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. The presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.

  1. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself

    National Research Council Canada - National Science Library

    McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  2. Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself: e0118187

    National Research Council Canada - National Science Library

    Cian M McCrudden; Martin G O'Rourke; Kim E Cherry; Hiu-Fung Yuen; Declan O'Rourke; Muhammad Babur; Brian A Telfer; Huw D Thomas; Patrick Keane; Thiagarajan Nambirajan; Chris Hagan; Joe M O'Sullivan; Chris Shaw; Kaye J Williams; Nicola J Curtin; David G Hirst; Tracy Robson

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  3. Changes in conformation of myosin heads during the development of isometric contraction and rapid shortening in single frog muscle fibres.

    Science.gov (United States)

    Piazzesi, G; Reconditi, M; Dobbie, I; Linari, M; Boesecke, P; Diat, O; Irving, M; Lombardi, V

    1999-01-15

    1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

  4. Changes in the myosin secondary structure and shrimp surimi gel strength induced by dense phase carbon dioxide.

    Science.gov (United States)

    Guo, Minghui; Liu, Shucheng; Ismail, Marliya; Farid, Mohammed M; Ji, Hongwu; Mao, Weijie; Gao, Jing; Li, Chengyong

    2017-07-15

    Dense phase carbon dioxide (DPCD) could induce protein conformation changes. Myosin and shrimp surimi from Litopenaeus vannamei were treated with DPCD at 5-25MPa and 40-60°C for 20min. Myosin secondary structure was investigated by circular dichroism and shrimp surimi gel strength was determined using textural analysis to develop correlations between them. DPCD had a greater effect on secondary structure and gel strength than heating. With increasing pressure and temperature, the α-helix content of DPCD-treated myosin decreased, while the β-sheet, β-turn and random coil contents increased, and the shrimp surimi gel strength increased. The α-helix content was negatively correlated with gel strength, while the β-sheet, β-turn and random coil contents were positively correlated with gel strength. Therefore, when DPCD induced myosin to form a gel, the α-helix of myosin was unfolded and gradually converted to a β-sheet. Such transformations led to protein-protein interactions and cross-linking, which formed a three-dimensional network to enhance the gel strength.

  5. Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring.

    Science.gov (United States)

    Giordanengo, L; Maldonado, C; Rivarola, H W; Iosa, D; Girones, N; Fresno, M; Gea, S

    2000-11-01

    Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

  6. Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution

    Science.gov (United States)

    Hu, Zhongjun; Taylor, Dianne W.; Reedy, Michael K.; Edwards, Robert J.; Taylor, Kenneth A.

    2016-01-01

    We describe a cryo–electron microscopy three-dimensional image reconstruction of relaxed myosin II–containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin’s long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for Lethocerus. Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation. PMID:27704041

  7. Effects of FSGS-associated mutations on the stability and function of myosin-1 in fission yeast

    Directory of Open Access Journals (Sweden)

    Jing Bi

    2015-08-01

    Full Text Available Point mutations in the human MYO1E gene, encoding class I myosin Myo1e, are associated with focal segmental glomerulosclerosis (FSGS, a primary kidney disorder that leads to end-stage kidney disease. In this study, we used a simple model organism, fission yeast Schizosaccharomyces pombe, to test the effects of FSGS-associated mutations on myosin activity. Fission yeast has only one class I myosin, Myo1, which is involved in actin patch assembly at the sites of endocytosis. The amino acid residues mutated in individuals with FSGS are conserved between human Myo1e and yeast Myo1, which allowed us to introduce equivalent mutations into yeast myosin and use the resulting mutant strains for functional analysis. Yeast strains expressing mutant Myo1 exhibited defects in growth and endocytosis similar to those observed in the myo1 deletion strain. These mutations also disrupted Myo1 localization to endocytic actin patches and resulted in mis-localization of Myo1 to eisosomes, linear membrane microdomains found in yeast cells. Although both mutants examined in this study exhibited loss of function, one of these mutants was also characterized by the decreased protein stability. Thus, using the yeast model system, we were able to determine that the kidney-disease-associated mutations impair myosin functional activity and have differential effects on protein stability.

  8. Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.

    Science.gov (United States)

    Boëda, Batiste; El-Amraoui, Aziz; Bahloul, Amel; Goodyear, Richard; Daviet, Laurent; Blanchard, Stéphane; Perfettini, Isabelle; Fath, Karl R; Shorte, Spencer; Reiners, Jan; Houdusse, Anne; Legrain, Pierre; Wolfrum, Uwe; Richardson, Guy; Petit, Christine

    2002-12-16

    Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.

  9. Alternative exon-encoding regions of Locusta migratoria muscle myosin modulate the pH dependence of ATPase activity.

    Science.gov (United States)

    Li, J; Lu, Z; He, J; Chen, Q; Wang, X; Kang, L; Li, X-D

    2016-12-01

    Whereas the vertebrate muscle myosin heavy chains (MHCs) are encoded by a family of Mhc genes, most insects examined to date contain a single Mhc gene and produce all of the different MHC isoforms by alternative RNA splicing. Here, we found that the migratory locust, Locusta migratoria, has one Mhc gene, which contains 41 exons, including five alternative exclusive exons and one differently included penultimate exon, and potentially encodes 360 MHC isoforms. From the adult L. migratoria, we identified 14 MHC isoforms (including two identical isoforms): four from flight muscle (the thorax dorsal longitudinal muscle), three from jump muscle (the hind leg extensor tibiae muscle) and seven from the abdominal intersegmental muscle. We purified myosins from flight muscle and jump muscle and characterized their motor activities. At neutral pH, the flight and the jump muscle myosins displayed similar levels of in vitro actin-gliding activity, whereas the former had a slightly higher actin-activated ATPase activity than the latter. Interestingly, the pH dependences of the actin-activated ATPase activity of these two myosins are different. Because the dominant MHC isoforms in these two muscles are identical except for the two alternative exon-encoding regions, we propose that these two alternative regions modulate the pH dependence of L. migratoria muscle myosin.

  10. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Tsuda, Takashi; Izumi, Tohru; Shibata, Akira (Niigata Univ. (Japan). School of Medicine)

    1992-08-01

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51[+-]11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of [sup 201]Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author).

  11. Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.

    Science.gov (United States)

    Previs, Michael J; Mun, Ji Young; Michalek, Arthur J; Previs, Samantha Beck; Gulick, James; Robbins, Jeffrey; Warshaw, David M; Craig, Roger

    2016-03-22

    During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.

  12. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    Science.gov (United States)

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  13. Allosteric communication in myosin V: from small conformational changes to large directed movements.

    Directory of Open Access Journals (Sweden)

    M Cecchini

    Full Text Available The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

  14. Myosin content of single muscle fibers following short-term disuse and active recovery in young and old healthy men

    DEFF Research Database (Denmark)

    Hvid, Lars G; Brocca, Lorenza; Ørtenblad, Niels

    2017-01-01

    Short-term disuse and subsequent recovery affect whole muscle and single myofiber contractile function in young and old. While the loss and recovery of single myofiber specific force (SF) following disuse and rehabilitation has been shown to correlate with alterations in myosin concentrations...... in young, it is unknown whether similar relationships exist in old. Therefore, the purpose of the present study was to examine the effect of 14days lower limb disuse followed by 28days of active recovery on single muscle fiber myosin content in old (68yrs) and young (24yrs) recreationally physically active...... contractile capacity of MHC 2a fibers. In conclusion, adaptive changes in myofiber myosin content appear to occur rapidly following brief periods of disuse (2wks) and after subsequent active recovery (4wks) in young and old, which contribute to alterations in contractile function at the single muscle fiber...

  15. The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II.

    Science.gov (United States)

    Rujano, Maria A; Sanchez-Pulido, Luis; Pennetier, Carole; le Dez, Gaelle; Basto, Renata

    2013-11-01

    Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. Here, we characterized the function of the Drosophila ASPM orthologue, Asp, and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. We show that size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. We propose that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness.

  16. Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To study the effect of 4-6 weeks' treadmill training of male SD rats on the contractile function of their gastrocnemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4-6 weeks at an intensity of about 75% VO2max (18.5-24 m/min,gradient of 0°,each training session lasting 50 minutes,twice a day). The content of gastrocnemius MHC mRNA was tested by rever...

  17. Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

    Directory of Open Access Journals (Sweden)

    J A Zoladz

    2004-10-01

    Full Text Available Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A, national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training. Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC composition. Significant differences (P<0.05 regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX. Unexpectedly, endurance athletes (group B such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A. We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

  18. Botulinum Toxin Type A Inhibits α-Smooth Muscle Actin and Myosin II Expression in Fibroblasts Derived From Scar Contracture.

    Science.gov (United States)

    Chen, Minliang; Yan, Tongtong; Ma, Kui; Lai, Linying; Liu, Chang; Liang, Liming; Fu, Xiaobing

    2016-09-01

    Scar contracture (SC) is one of the most common complications resulting from major burn injuries. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Recent reports suggest that botulinum toxin type A (BTXA) is effective at reducing SC clinically, but the molecular mechanism for this action is unknown. α-Smooth muscle actin (α-SMA) and myosin II are the main components of stress fibers, which are the contractile structures of fibroblasts. The effects of BTXA on α-SMA and myosin II in SC are still unknown. This study aimed to explore the effect of BTXA on α-SMA and myosin II expression in fibroblasts derived from SC and to elucidate its actual mechanism further. Fibroblasts were isolated from tissue specimens of SC. Fibroblasts were cultured in Dulbecco modified Eagle medium with different concentrations of BTXA and their proliferation was analyzed through the tetrazolium-based colorimetric method at 1, 4, and 7 days. Proteins of α-SMA and myosin II were checked using Western blot in fibroblasts treated with different concentrations of BTXA at 1, 4, and 7 days. Fibroblasts without BTXA treatment had a higher proliferation than that in other groups, which indicated that the proliferation of fibroblasts was significantly inhibited by BTXA (P < 0.05). Proteins of α-SMA and myosin II between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (P < 0.05). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from SC and reduced the expression of α-SMA and myosin II, which provided theoretical support for the application of BTXA to control SC.

  19. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, S F; Hoffmann, E K

    2002-01-01

    Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization....... In contrast, the previously reported shrinkage-induced actin polymerization [Pedersen et al. (1999) Exp. Cell Res. 252, 63-74] was independent of Rho kinase, p38, myosin light chain kinase (MLCK), and protein kinase C (PKC), which thus do not exert their effects on the shrinkage-activated transporters via...... by osmotic shrinkage and by the serine/threonine phosphatase inhibitor Calyculin A (CL-A). Both stimuli caused Rho kinase-dependent myosin II relocation to the cortical cytoplasm, but in contrast to the shrinkage-induced F-actin polymerization, CL-A treatment elicited a slight F-actin depolymerization...

  20. The Elastic Properties of the Structurally Characterized Myosin II S2 Subdomain: A Molecular Dynamics and Normal Mode Analysis

    OpenAIRE

    2008-01-01

    The elastic properties (stretching and bending moduli) of myosin are expected to play an important role in its function. Of particular interest is the extended α-helical coiled-coil portion of the molecule. Since there is no high resolution structure for the entire coiled-coil, a study is made of the scallop myosin II S2 subdomain for which an x-ray structure is available (Protein Data Bank 1nkn). We estimate the stretching and bending moduli of the S2 subdomain with an atomic level model by ...

  1. X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers

    Science.gov (United States)

    Kimura, Masako; Li, Zhao-bo; Ohno, Tetsuo; Takemori, Shigeru; Hoh, Joseph F. Y.; Yagi, Naoto

    2016-01-01

    The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca2+] = 10−6.8 M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm−1 along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments. PMID:26911280

  2. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    Directory of Open Access Journals (Sweden)

    Saroj Kumar

    Full Text Available Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1. The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

  3. Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age.

    Science.gov (United States)

    Belik, J; Kerc, Ewa; Pato, Mary D

    2006-03-01

    We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

  4. The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L

    Science.gov (United States)

    Assis, L. H. P.; Silva-Junior, R. M. P.; Dolce, L. G.; Alborghetti, M. R.; Honorato, R. V.; Nascimento, A. F. Z.; Melo-Hanchuk, T. D.; Trindade, D. M.; Tonoli, C. C. C.; Santos, C. T.; Oliveira, P. S. L.; Larson, R. E.; Kobarg, J.; Espreafico, E. M.; Giuseppe, P. O.; Murakami, M. T.

    2017-01-01

    Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes. PMID:28266547

  5. The zebrafish goosepimples/myosin Vb mutant exhibits cellular attributes of human microvillus inclusion disease.

    Science.gov (United States)

    Sidhaye, Jaydeep; Pinto, Clyde Savio; Dharap, Shweta; Jacob, Tressa; Bhargava, Shobha; Sonawane, Mahendra

    2016-11-01

    Microvillus inclusion disease (MVID) is a life-threatening enteropathy characterised by malabsorption and incapacitating fluid loss due to chronic diarrhoea. Histological analysis has revealed that enterocytes in MVID patients exhibit reduction of microvilli, presence of microvillus inclusion bodies and intestinal villus atrophy, whereas genetic linkage analysis has identified mutations in myosin Vb gene as the main cause of MVID. In order to understand the cellular basis of MVID and the associated formation of inclusion bodies, an animal model that develops ex utero and is tractable genetically as well as by microscopy would be highly useful. Here we report that the intestine of the zebrafish goosepimples (gsp)/myosin Vb (myoVb) mutant shows severe reduction in intestinal folds - structures similar to mammalian villi. The loss of folds is further correlated with changes in the shape of enterocytes. In striking similarity with MVID patients, zebrafish gsp/myoVb mutant larvae exhibit microvillus atrophy, microvillus inclusions and accumulation of secretory material in enterocytes. We propose that the zebrafish gsp/myoVb mutant is a valuable model to study the pathophysiology of MVID. Furthermore, owing to the advantages of zebrafish in screening libraries of small molecules, the gsp mutant will be an ideal tool to identify compounds having therapeutic value against MVID.

  6. Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

    Science.gov (United States)

    Andrecka, Joanna; Ortega Arroyo, Jaime; Takagi, Yasuharu; de Wit, Gabrielle; Fineberg, Adam; MacKinnon, Lachlan; Young, Gavin; Sellers, James R; Kukura, Philipp

    2015-01-01

    Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps. DOI: http://dx.doi.org/10.7554/eLife.05413.001 PMID:25748137

  7. Cytoskeletal turnover and Myosin contractility drive cell autonomous oscillations in a model of Drosophila Dorsal Closure

    Science.gov (United States)

    Machado, P. F.; Blanchard, G. B.; Duque, J.; Gorfinkiel, N.

    2014-06-01

    Oscillatory behaviour in force-generating systems is a pervasive phenomenon in cell biology. In this work, we investigate how oscillations in the actomyosin cytoskeleton drive cell shape changes during the process of Dorsal Closure (DC), a morphogenetic event in Drosophila embryo development whereby epidermal continuity is generated through the pulsatile apical area reduction of cells constituting the amnioserosa (AS) tissue. We present a theoretical model of AS cell dynamics by which the oscillatory behaviour arises due to a coupling between active myosin-driven forces, actin turnover and cell deformation. Oscillations in our model are cell-autonomous and are modulated by neighbour coupling, and our model accurately reproduces the oscillatory dynamics of AS cells and their amplitude and frequency evolution. A key prediction arising from our model is that the rate of actin turnover and Myosin contractile force must increase during DC in order to reproduce the decrease in amplitude and period of cell area oscillations observed in vivo. This prediction opens up new ways to think about the molecular underpinnings of AS cell oscillations and their link to net tissue contraction and suggests the form of future experimental measurements.

  8. Myosin binding protein C:Structural abnormalities in familial hypertrophic cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    The muscle protein myosin binding protein C (MyBPC) is a large multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Mutations in MyBPC are strongly associated with the heart disease familial hypertrophic cardiomyopathy (FHC) and these experiments of nature have provided some insight into the intricate workings of this protein in the heart. While some regions of the MyBPC molecule have been assigned a function in the regulation of muscle contraction, the interaction of other regions with various parts of the myosin molecule and the sarcomeric proteins, actin and titin, remain obscure. In additic n, several intra-domain interactions between adjacent MyBPC molecules have been identified. Although the basic structure of the molecule (a series of immunoglobulin and fibronectin domains) has been elucidated, the assembly of MyBPC in the sarcomere is a topic for debate. By analysing the MyBPC sequence with respect to FHC-causing mutations it is possible to identify individual residues or regions of each domain that may be important either for binding or regulation. This review looks at the current literature, in concert with alignments and the structural models of MyBPC, in an attempt to understand how FHC mutations may lead to the disease state.

  9. Impact of resistance exercise during bed rest on skeletal muscle sarcopenia and myosin isoform distribution

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Feeback, D. L.; Talmadge, R. J.; Stevens, B. R.; Lieberman, S. A.; Greenisen, M. C.

    1998-01-01

    Because resistance exercise (REx) and bed-rest unloading (BRU) are associated with opposing adaptations, our purpose was to test the efficacy of REx against the effects of 14 days of BRU on the knee-extensor muscle group. Sixteen healthy men were randomly assigned to no exercise (NoEx; n = 8) or REx (n = 8). REx performed five sets of leg press exercise with 80-85% of one repetition maximum (1 RM) every other day during BRU. Muscle samples were removed from the vastus lateralis muscle by percutaneous needle biopsy. Myofiber distribution was determined immunohistochemically with three monoclonal antibodies against myosin heavy chain (MHC) isoforms (I, IIa, IIx). MHC distribution was further assessed by quantitative gel electrophoresis. Dynamic 1-RM leg press and unilateral maximum voluntary isometric contraction (MVC) were determined. Maximal neural activation (root mean squared electromyogram) and rate of torque development (RTD) were measured during MVC. Reductions (P training-specific strength. Unlike spaceflight, BRU did not induce shifts in myosin phenotype. The reported benefits of REx may prove useful in prescribing exercise for astronauts in microgravity.

  10. ENDOR and ELDOR studies of x-irradiated polycrystalline dipeptides, myosin, and actomyosin

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, J.S. (Univ. of Alabama, Tuscaloosa); Dickinson, A.C.; Kispert, L.D.

    1979-12-27

    ENDOR and ELDOR studies have been carried out for nine dipeptide powders as well as powders of myosin and actomyosin x ray irradiated at 77/sup 0/K in an attempt to characterize the final radical stable upon annealing between 183 and 260/sup 0/K. The dipeptides studied were glycylglycine, L-alanylglycine, glycyl-L-alanine, L-alanyl-L-alanine, glycyl-L-aspartic acid, glycyl-L-glutamic acid, glycyl-L-methionine, glycyl-L-serine, and L-lysyl-L-lysine. Nitrogen ENDOR spectra have been observed between 1 and 8 MHz for each powder and the nitrogen hyperfine and quadrupole tensor has been estimated. Analysis of the ENDOR, ELDOR, and ESR spectra indicates at least one of the final radicals in the dipeptide powders (except Gly-Gly, and possibly Gly-Glu, Gly-Ser) to be the decarboxylation product NH/sub 2/CHRCONHCHR' rather than just the abstraction type (NH/sub 3//sup +/-CHRONHCR'COO/sup -/) previously identified in irradiated dipeptide ices. A decarboxylation type radical is also present as a final radical in the irradiated myosin and actomyosin.

  11. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  12. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    Science.gov (United States)

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  13. The role of myosin II in glioma invasion: A mathematical model

    Science.gov (United States)

    Lee, Wanho; Lim, Sookkyung; Kim, Yangjin

    2017-01-01

    Gliomas are malignant tumors that are commonly observed in primary brain cancer. Glioma cells migrate through a dense network of normal cells in microenvironment and spread long distances within brain. In this paper we present a two-dimensional multiscale model in which a glioma cell is surrounded by normal cells and its migration is controlled by cell-mechanical components in the microenvironment via the regulation of myosin II in response to chemoattractants. Our simulation results show that the myosin II plays a key role in the deformation of the cell nucleus as the glioma cell passes through the narrow intercellular space smaller than its nuclear diameter. We also demonstrate that the coordination of biochemical and mechanical components within the cell enables a glioma cell to take the mode of amoeboid migration. This study sheds lights on the understanding of glioma infiltration through the narrow intercellular spaces and may provide a potential approach for the development of anti-invasion strategies via the injection of chemoattractants for localization. PMID:28166231

  14. A Novel Myosin Essential Light Chain Mutation Causes Hypertrophic Cardiomyopathy with Late Onset and Low Expressivity

    Directory of Open Access Journals (Sweden)

    Paal Skytt Andersen

    2012-01-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is caused by mutations in genes encoding sarcomere proteins. Mutations in MYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT 21 mm, a resting left ventricular outflow gradient of 36 mm Hg, and left atrial dilation (54 mm. Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, in MYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion, MYL3 mutations can present with low expressivity and late onset.

  15. Reversal of the Myosin Power Stroke Induced by Fast Stretching of Intact Skeletal Muscle Fibers

    Science.gov (United States)

    Colombini, Barbara; Nocella, Marta; Benelli, Giulia; Cecchi, Giovanni; Griffiths, Peter J.; Bagni, M. Angela

    2009-01-01

    Abstract Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15–20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment. PMID:19948121

  16. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

    Science.gov (United States)

    Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

    2014-01-01

    Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

  17. [Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis].

    Science.gov (United States)

    Miuge, N S; Tikhonov, A V; Ozerniuk, N D

    2005-01-01

    mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

  18. Model of myosin node aggregation into a contractile ring: the effect of local alignment

    Energy Technology Data Exchange (ETDEWEB)

    Ojkic, Nikola; Vavylonis, Dimitrios [Department of Physics, Lehigh University, Bethlehem, PA 18015 (United States); Wu Jianqiu, E-mail: vavylonis@lehigh.edu [Department of Molecular Genetics and Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH 43210 (United States)

    2011-09-21

    Actomyosin bundles frequently form through aggregation of membrane-bound myosin clusters. One such example is the formation of the contractile ring in fission yeast from a broad band of cortical nodes. Nodes are macromolecular complexes containing several dozens of myosin-II molecules and a few formin dimers. The condensation of a broad band of nodes into the contractile ring has been previously described by a search, capture, pull and release (SCPR) model. In SCPR, a random search process mediated by actin filaments nucleated by formins leads to transient actomyosin connections among nodes that pull one another into a ring. The SCPR model reproduces the transport of nodes over long distances and predicts observed clump-formation instabilities in mutants. However, the model does not generate transient linear elements and meshwork structures as observed in some wild-type and mutant cells during ring assembly. As a minimal model of node alignment, we added short-range aligning forces to the SCPR model representing currently unresolved mechanisms that may involve structural components, cross-linking and bundling proteins. We studied the effect of the local node alignment mechanism on ring formation numerically. We varied the new parameters and found viable rings for a realistic range of values. Morphologically, transient structures that form during ring assembly resemble those observed in experiments with wild-type and cdc25-22 cells. Our work supports a hierarchical process of ring self-organization involving components drawn together from distant parts of the cell followed by progressive stabilization.

  19. Myosin head orientation: a structural determinant for the Frank-Starling relationship.

    Science.gov (United States)

    Farman, Gerrie P; Gore, David; Allen, Edward; Schoenfelt, Kelly; Irving, Thomas C; de Tombe, Pieter P

    2011-06-01

    The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I(1,1)/I(1,0) intensity ratio decreases, whereas the M3 meridional reflection intensity (I(M3)) increases, concomitant with increases in diastolic and systolic force. Using a short (∼10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by I(M3)) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.

  20. Myosin head orientation: a structural determinant for the Frank-Starling relationship

    Energy Technology Data Exchange (ETDEWEB)

    Farman, Gerrie P.; Gore, David; Allen, Edward; Schoenfelt, Kelly; Irving, Thomas C.; de Tombe, Pieter P. (IIT); (UIC)

    2011-09-06

    The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I{sub 1,1}/I{sub 1,0} intensity ratio decreases, whereas the M3 meridional reflection intensity (I{sub M3}) increases, concomitant with increases in diastolic and systolic force. Using a short ({approx}10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by IM3) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.

  1. Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

    Directory of Open Access Journals (Sweden)

    Kanji Oshima

    Full Text Available The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation

  2. An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

    NARCIS (Netherlands)

    Schneeberger, Kerstin; Vogel, Georg F; Teunissen, Hans; van Ommen, Domenique D; Begthel, Harry; El Bouazzaoui, Layla; van Vugt, Anke H M; Beekman, Jeffrey M; Klumperman, Judith; Müller, Thomas; Janecke, Andreas; Gerner, Patrick; Huber, Lukas A; Hess, Michael W; Clevers, Hans; van Es, Johan H; Nieuwenhuis, Edward E S; Middendorp, Sabine

    2015-01-01

    Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of

  3. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation

    Directory of Open Access Journals (Sweden)

    Kate Fisher

    2013-10-01

    Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

  4. Myosin IXB gene region and gluten intolerance : linkage to coeliac disease and a putative dermatitis herpetiformis association

    NARCIS (Netherlands)

    Koskinen, L. L. E.; Korponay-Szabo, I. R.; Viiri, K.; Juuti-Uusitalo, K.; Kaukinen, K.; Lindfors, K.; Mustalahti, K.; Kurppa, K.; Adany, R.; Pocsai, Z.; Szeles, G.; Einarsdottir, E.; Wijmenga, C.; Maeki, M.; Partanen, J.; Kere, J.; Saavalainen, P.

    2008-01-01

    Background: Coeliac disease is caused by dietary gluten, which triggers chronic inflammation of the small intestine in genetically predisposed individuals. In one quarter of the patients the disease manifests in the skin as dermatitis herpetiformis. Recently, a novel candidate gene, myosin IXB on ch

  5. Harmonic force spectroscopy reveals a force-velocity curve from a single human beta cardiac myosin motor

    Science.gov (United States)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian; Mortensen, Kim; Flyvbjerg, Henrik; Spudich, James

    2014-03-01

    A muscle contracts rapidly under low load, but slowly under high load. Its molecular mechanisms remain to be elucidated, however. During contraction, myosins in thick filaments interact with actin in thin filaments in the sarcomere, cycling between a strongly bound (force producing) state and a weakly bound (relaxed) state. Huxley et al. have previously proposed that the transition from the strong to the weak interaction can be modulated by a load. We use a new method we call ``harmonic force spectroscopy'' to extract a load-velocity curve from a single human beta cardiac myosin II motor. With a dual-beam optical trap, we hold an actin dumbbell over a myosin molecule anchored to the microscope stage that oscillates sinusoidally. Upon binding, the motor experiences an oscillatory load with a mean that is directed forward or backward, depending on binding location We find that the bound time at saturating [ATP] is exponentially correlated with the mean load, which is explained by Arrhenius transition theory. With a stroke size measurement, we obtained a load-velocity curve from a single myosin. We compare the curves for wild-type motors with mutants that cause hypertrophic cardiomyopathies, to understand the effects on the contractile cycle

  6. An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

    NARCIS (Netherlands)

    Schneeberger, Kerstin; Vogel, Georg F.; Teunissen, Hans; Zomer-van Ommen, Domenique; Begthel, Harry; El Bouazzaoui, Layla; van Vugt, Anke H. M.; Beekman, JM; Klumperman, Judith; Mueller, Thomas; Janecke, Andreas; Gerner, Patrick; Huber, Lukas A.; Hess, Michael W.; Clevers, Hans; van Es, Johan H.; Nieuwenhuis, Edward E. S.; Middendorp, Sabine

    2015-01-01

    Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of M

  7. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States); Bernstein, Sanford I., E-mail: sbernst@sciences.sdsu.edu [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States)

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  8. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Hosaka, Toshio [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Teshigawara, Kiyoshi [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Funaki, Makoto, E-mail: m-funaki@clin.med.tokushima-u.ac.jp [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  9. Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

    Directory of Open Access Journals (Sweden)

    Tolliday Nicola J

    2006-06-01

    Full Text Available Abstract Background All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae, in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD of budding yeast Myo1. Results We show that the MLD is a small region in the centre of the tail of Myo1 and that it is both necessary and sufficient for localisation of Myo1 to the yeast bud neck, the pre-determined site of cell division. Hydrodynamic measurements of the MLD, purified from bacteria or yeast, show that it is likely to exist as a trimer. We also examine the importance of a small region of low coiled coil forming probability within the MLD, which we call the hinge region. Removal of the hinge region prevents contraction of the CR. Using fluorescence recovery after photobleaching (FRAP, we show that GFP-tagged MLD is slightly more dynamic than the GFP-tagged full length molecule but less dynamic than the GFP-tagged Myo1 construct lacking the hinge region. Conclusion Our results define the intrinsic determinant for the localization of budding yeast myosin II and show

  10. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    Science.gov (United States)

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2013-01-01

    Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi. PMID:24187546

  11. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

    Science.gov (United States)

    Suggs, Jennifer A; Melkani, Girish C; Glasheen, Bernadette M; Detor, Mia M; Melkani, Anju; Marsan, Nathan P; Swank, Douglas M; Bernstein, Sanford I

    2017-06-01

    Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro

  12. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    Directory of Open Access Journals (Sweden)

    Amirali eSattarzadeh

    2013-10-01

    Full Text Available Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the PAL sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi.

  13. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects

    Directory of Open Access Journals (Sweden)

    Jennifer A. Suggs

    2017-06-01

    Full Text Available Individuals with inclusion body myopathy type 3 (IBM3 display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in

  14. Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform

    Directory of Open Access Journals (Sweden)

    Yi Ting

    2012-11-01

    Full Text Available Abstract It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG and that exposure of zinc ion (Zn2+ to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our

  15. Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform.

    Science.gov (United States)

    Yi, Ting; Cheema, Yaser; Tremble, Sarah M; Bell, Stephen P; Chen, Zengyi; Subramanian, Meenakumari; LeWinter, Martin M; VanBuren, Peter; Palmer, Bradley M

    2012-11-02

    It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the

  16. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    Science.gov (United States)

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

  17. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

    Science.gov (United States)

    Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

    2012-06-01

    Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

  18. The Intensity Of The 2.7nm Reflection As A Constraint For Models Of Myosin Docking To Actin

    Energy Technology Data Exchange (ETDEWEB)

    Reconditi, Massimo; Irving, Tom C.; (IIT); (U.Florence)

    2009-03-16

    Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data (Mendelson and Morris, 1997 PNAS 94:8533; Holmes et al. 2003 Nature 425:423). We have used data from small angle X-ray fiber diffraction from living muscle to check the predictions of these models. Whole sartorius muscles from Rana pipiens were mounted in a chamber containing Ringer's solution at 10 C and at rest length at the BioCAT beamline (18 ID, Advanced Photon Source, Argonne, IL-U.S.A.). The muscles were activated by electrical stimulation and the force was recorded with a muscle lever system type 300B (Aurora Scientific). X-ray patterns were collected with 1s total exposures at rest and during isometric contraction out to 0.5 nm{sup -1} in reciprocal space, as the higher angle reflections are expected to be more sensitive to the arrangement of myosin heads on actin. We observed that during isometric contraction the meridional reflection originating from the 2.73nm repeat of the actin monomers along the actin filament increases its intensity by a factor 2.1 {+-} 0.2 relative to rest. Among the models tested, Holmes et al. fits the data when the actin filament is decorated with 30-40% the total available myosin heads, a fraction similar to that estimated with fast single fiber mechanics by Piazzesi et al. (2007, Cell 131:784). However, when the mismatch between the periodicities of actin and myosin filaments is taken into account, none of the models can reproduce the fiber diffraction data. We suggest that the fiber diffraction data should be used as a further constraint on new high resolution models for the docking of the myosin heads on actin.

  19. Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

    Science.gov (United States)

    Prochniewicz, Ewa; Guhathakurta, Piyali; Thomas, David D.

    2013-01-01

    We have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, in order to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final anisotropy cooperatively and to a similar extent, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of saturating ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin. PMID:23339370

  20. Small heat shock protein Hsp27 protects myosin S1 from heat-induced aggregation, but not from thermal denaturation and ATPase inactivation.

    Science.gov (United States)

    Markov, Denis I; Pivovarova, Anastasia V; Chernik, Ivan S; Gusev, Nikolai B; Levitsky, Dmitrii I

    2008-04-30

    We applied different methods, such as turbidity measurements, dynamic light scattering, differential scanning calorimetry and co-sedimentation assay, to analyze the interaction of small heat shock protein Hsp27 with isolated myosin head (myosin subfragment 1, S1) under heat-stress conditions. Upon heating at 43 degrees C, Hsp27 effectively suppresses S1 aggregation, and this effect is enhanced by mutations mimicking Hsp27 phosphorylation. However, Hsp27 was unable to prevent thermal unfolding of myosin heads and to maintain their ATPase activity under heat-shock conditions.

  1. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  2. Apical domain polarization localizes actin-myosin activity to drive ratchet-like apical constriction.

    Science.gov (United States)

    Mason, Frank M; Tworoger, Michael; Martin, Adam C

    2013-08-01

    Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

  3. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    Science.gov (United States)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  4. Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor.

    Science.gov (United States)

    Lartey, Jon; Taggart, Julie; Robson, Stephen; Taggart, Michael

    2016-01-01

    Myosin light-chain phosphatase is a trimeric protein that hydrolyses phosphorylated myosin II light chains (MYLII) to cause relaxation in smooth muscle cells including those of the uterus. A major component of the phosphatase is the myosin targeting subunit (MYPT), which directs a catalytic subunit to dephosphorylate MYLII. There are 5 main MYPT family members (MYPT1 (PPP1R12A), MYPT2 (PPP1R12B), MYPT3 (PPP1R16A), myosin binding subunit 85 MBS85 (PPP1R12C) and TIMAP (TGF-beta-inhibited membrane-associated protein (PPP1R16B)). Nitric oxide (NO)-mediated smooth muscle relaxation has in part been attributed to activation of the phosphatase by PKG binding to a leucine zipper (LZ) dimerization domain located at the carboxyl-terminus of PPP1R12A. In animal studies, alternative splicing of PPP1R12A can lead to the inclusion of a 31-nucleotide exonic segment that generates a LZ negative (LZ-) isovariant rendering the phosphatase less sensitive to NO vasodilators and alterations in PPP1R12ALZ- and LZ+ expression have been linked to phenotypic changes in smooth muscle function. Moreover, PPP1R12B and PPP1R12C, but not PPP1R16A or PPP1R16B, have the potential for LZ+/LZ- alternative splicing. Yet, by comparison to animal studies, the information on human MYPT genomic sequences/mRNA expressions is scant. As uterine smooth muscle undergoes substantial remodeling during pregnancy we were interested in establishing the patterns of expression of human MYPT isovariants during this process and also following labor onset as this could have important implications for determining successful pregnancy outcome. We used cross-species genome alignment, to infer putative human sequences not available in the public domain, and isovariant-specific quantitative PCR, to analyse the expression of mRNA encoding putative LZ+ and LZ- forms of PPP1R12A, PPP1R12B and PPP1R12C as well as canonical PPP1R16A and PPP1R16B genes in human uterine smooth muscle from non-pregnant, pregnant and in

  5. Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

    Science.gov (United States)

    Sung, Jongmin; Nag, Suman; Mortensen, Kim I.; Vestergaard, Christian L.; Sutton, Shirley; Ruppel, Kathleen; Flyvbjerg, Henrik; Spudich, James A.

    2015-08-01

    Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using `harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

  6. Actin- and Myosin-Dependent Vesicle Loading of Presynaptic Docking Sites Prior to Exocytosis.

    Science.gov (United States)

    Miki, Takafumi; Malagon, Gerardo; Pulido, Camila; Llano, Isabel; Neher, Erwin; Marty, Alain

    2016-08-17

    Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    Science.gov (United States)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  8. Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations.

    Directory of Open Access Journals (Sweden)

    Haibo Yu

    2007-02-01

    Full Text Available Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP, the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide

  9. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    Science.gov (United States)

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts.

  10. Determination of the critical residues responsible for cardiac myosin binding protein C's interactions.

    Science.gov (United States)

    Bhuiyan, Md Shenuarin; Gulick, James; Osinska, Hanna; Gupta, Manish; Robbins, Jeffrey

    2012-12-01

    Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.

  11. The Intriguing Dual Lattices of the Myosin Filaments in Vertebrate Striated Muscles: Evolution and Advantage

    Directory of Open Access Journals (Sweden)

    Pradeep K. Luther

    2014-12-01

    Full Text Available Myosin filaments in vertebrate striated muscle have a long roughly cylindrical backbone with cross-bridge projections on the surfaces of both halves except for a short central bare zone. In the middle of this central region the filaments are cross-linked by the M-band which holds them in a well-defined hexagonal lattice in the muscle A-band. During muscular contraction the M-band-defined rotation of the myosin filaments around their long axes influences the interactions that the cross-bridges can make with the neighbouring actin filaments. We can visualise this filament rotation by electron microscopy of thin cross-sections in the bare-region immediately adjacent to the M-band where the filament profiles are distinctly triangular. In the muscles of teleost fishes, the thick filament triangular profiles have a single orientation giving what we call the simple lattice. In other vertebrates, for example all the tetrapods, the thick filaments have one of two orientations where the triangles point in opposite directions (they are rotated by 60° or 180° according to set rules. Such a distribution cannot be developed in an ordered fashion across a large 2D lattice, but there are small domains of superlattice such that the next-nearest neighbouring thick filaments often have the same orientation. We believe that this difference in the lattice forms can lead to different contractile behaviours. Here we provide a historical review, and when appropriate cite recent work related to the emergence of the simple and superlattice forms by examining the muscles of several species ranging back to primitive vertebrates and we discuss the functional differences that the two lattice forms may have.

  12. Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

    Science.gov (United States)

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Suita, Kenji; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2015-01-01

    Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

  13. 5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

    Institute of Scientific and Technical Information of China (English)

    YANG Chunxiang; WEI Dongmei; CHEN Chen; YU Weiping; ZHU Minsheng

    2005-01-01

    Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

  14. Shifts in the myosin heavy chain isozymes in the mouse heart result in increased energy efficiency

    Science.gov (United States)

    Hoyer, Kirsten; Krenz, Maike; Robbins, Jeffrey; Ingwall, Joanne S.

    2007-01-01

    Cardiac-specific transgenesis in the mouse is widely used to study the basic biology and chemistry of the heart and to model human cardiovascular disease. A fundamental difference between mouse and human hearts is the background motor protein: mouse hearts contain predominantly the αα-myosin heavy chain (MyHC) isozyme while human hearts contain predominantly the ββ-MyHC isozyme. Although the intrinsic differences in mechanical and enzymatic properties of the αα- and ββ-MyHC molecules are well known, the consequences of isozyme shifts on energetic of the intact beating heart remain unknown. Therefore, we compared the free energy of ATP hydrolysis (|ΔG~ATP|) determined by 31P NMR spectroscopy in isolated perfused littermate mouse hearts containing the same amount of myosin comprised of either >95% αα-MyHC or ~83% ββ-MyHC. |ΔG~ATP| was ~2 kJ mol−1 higher in the ββ-MyHC hearts at all workloads. Furthermore, upon inotropic challenge, hearts containing predominantly ββ-MyHC hearts increased developed pressure more than αα-MyHC hearts whereas heart rate increased more in αα-MyHC hearts. Thus, hearts containing predominantly the ββ-MyHC isozyme are more energy efficient than αα-MyHC hearts. We suggest that these fundamental differences in the motor protein energy efficiency at the whole heart level should be considered when interpreting results using mouse-based cardiovascular modeling of normal and diseased human heart. PMID:17054980

  15. Dilated cardiomyopathy mutation (R134W in mouse cardiac troponin T induces greater contractile deficits against α-myosin heavy chain than against β-myosin heavy chain

    Directory of Open Access Journals (Sweden)

    Sampath K Gollapudi

    2016-10-01

    Full Text Available Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM in humans. However, it remains unclear whether a single determinant — such as myofilament Ca2+ sensitivity — is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT, which is known to modulate crossbridge (XB recruitment dynamics in a myosin heavy chain (MHC-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W of the human DCM mutation (R131W, we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC and transgenic mice (β-MHC. TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units than in β-MHC fiber bundles (0.08 pCa units. Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

  16. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    Science.gov (United States)

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  17. Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V*

    Science.gov (United States)

    Preller, Matthias; Bauer, Stefanie; Adamek, Nancy; Fujita-Becker, Setsuko; Fedorov, Roman; Geeves, Michael A.; Manstein, Dietmar J.

    2011-01-01

    The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures. PMID:21841195

  18. Ki-67 is an independent indicator in non-muscle invasive bladder cancer (NMIBC); combination of EORTC risk scores and Ki-67 expression could improve the risk stratification of NMIBC.

    Science.gov (United States)

    Ding, Weihong; Gou, Yuancheng; Sun, Chuanyu; Xia, Guowei; Wang, Hong; Chen, Zhongqing; Tan, Jun; Xu, Ke; Qiang, Ding

    2014-01-01

    To prove the predicting role of Ki-67 expression and to demonstrate that the combination of European Organization for Research and Treatment of Cancer (EORTC) risk scores and Ki-67 staining status could improve the risk stratification in a large series of patients with non-muscle invasive bladder cancer (NMIBC). From October 2002 to July 2010, in our cohort, 332 patients who were treated with transurethral resection of the bladder tumor were diagnosed with NMIBC by histopathologic analysis. Two experienced uropathologists rereviewed the slides. The EORTC risk scores for recurrence and progression were determined. Ki-67 expression was evaluated using immunohistochemical studies and scored for intensity and area of staining. We correlated Ki-67 expression scores with clinical and pathologic variables. We evaluated the prognosis role of EORTC risk scores, Ki-67 staining, and their combination on tumor recurrence-free survival and progression-free survival (PFS) by univariate analysis, multivariate analysis, and Kaplan-Meier survival curves. With a median follow-up of 47 (range, 2-124) months, 119 patients (35.8%) had tumor recurrence and 40 patients (12%) had tumor progression. Ki-67 positivity (Ki-67>25%) was reported in 108 tumors (32.5%), and it was significantly associated with high EORTC risk scores for both tumor recurrence and progression. In univariate analysis, multifocality, tumor size, tumor stage, tumor grade, and Ki-67 staining correlated with recurrence-free survival, whereas tumor size, tumor stage, tumor grade, concomitant CIS, and Ki-67 staining correlated with PFS. In multivariable analysis, Ki-67 expression was an independent risk factor for predicting tumor recurrence (hazard ratio, 2.14; PEORTC risk scores and Ki-67 staining led to more accurate prediction for tumor recurrence and progression (log-rank test; PEORTC risk scores with Ki-67 expression could improve the risk stratification for both recurrence and progression in NMIBC. Copyright

  19. Alterations in ubiquitin ligase Siah-2 and its corepressor N-CoR after P-MAPA immunotherapy and anti-androgen therapy: new therapeutic opportunities for non-muscle invasive bladder cancer.

    Science.gov (United States)

    Garcia, Patrick Vianna; Apolinário, Letícia Montanholi; Böckelmann, Petra Karla; da Silva Nunes, Iseu; Duran, Nelson; Fávaro, Wagner José

    2015-01-01

    The present study describes the role of the ubiquitin ligase Siah-2 and corepressor N-CoR in controlling androgen receptor (AR) and estrogen receptors (ERα and ERβ) signaling in an appropriate animal model (Fischer 344 female rats) of non-muscle invasive bladder cancer (NMIBC), especially under conditions of anti-androgen therapy with flutamide. Furthermore, this study describes the mechanisms of a promising therapeutic alternative for NMIBC based on Protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) intravesical immunotherapy combined with flutamide, involving the interaction among steroid hormone receptors, their regulators and Toll-like receptors (TLRs). Our results demonstrated that increased Siah-2 and AR protein levels and decreased N-CoR, cytochrome P450 (CYP450) and estrogen receptors levels played a critical role in the urothelial carcinogenesis, probably leading to escape of urothelial cancer cells from immune system attack. P-MAPA immunotherapy led to distinct activation of innate immune system TLRs 2 and 4-mediated, resulting in increase of interferon signaling pathway, which was more effective in recovering the immunosuppressive tumor immune microenvironment and in recovering the bladder histology features than BCG (Bacillus Calmette-Guerin) treatments. The AR blockade therapy was important in the modulating of downstream molecules of TLR2 and TLR4 signaling pathway, decreasing the inflammatory cytokines signaling and enhancing the interferon signaling pathway when associated with P-MAPA. Taken together, the data obtained suggest that interferon signaling pathway activation and targeting AR and Siah-2 signals by P-MAPA intravesical immunotherapy alone and/ or in combination with AR blockade may provide novel therapeutic approaches for NMIBC.

  20. 添加非肌肉蛋白对鱼糜制品品质影响的初步研究%Research of Optimum Recipe of Surimi with Non-muscle Proteins

    Institute of Scientific and Technical Information of China (English)

    陈康; 戴志远; 王宏海; 翁丽萍

    2011-01-01

    T The effects of soybean protein isolation (SPI), gluten powder (GP) and whey protein concentration ( WPC ) on the qualities of surimi were investigated by texture analysis, color analysis and sensory evaluation. The resuits indicated that the addition of three kinds of non-muscle proteins increased the breaking force, gel strength, moisture holding and sensory, while it displayed opposite changes in deformation and whiteness. The optimum recipe (9.7% SPI,9.9% GP,7.6% WPC) was determined by surface response methodology (RSM), and the actual gel strength was (3 855. 012 ± 260. 682) g· mm under this condition.%实验采用质构分析法、色差分析法及感官评价法研究了大豆分离蛋白(SPI)、谷朊粉(GP)、乳清浓缩蛋白(WPC)对鱼糜制品品质的影响。结果表明:添加一定量的3种非肌肉蛋白可以明显地提高鱼糜制品的破断强度、凝胶强度、持水性和口感,但均降低了制品的凹陷深度和自度。以凝胶强度为优化值对3种非肌肉蛋白进行响应面优化后,确定了鱼糜制品的蛋白添加条件为:SPI9.7%、GP9.9%、WPC7.6%,实际凝胶强度为(3855.012±260.682)g·mm。

  1. The clinical course of non-muscle invasive bladder cancer after transuretral resection of the tumor with or without subsequent intravesical application of bacillus Calmette-Guérin: The influence of patients gender and age

    Directory of Open Access Journals (Sweden)

    Milošević Radovan

    2015-01-01

    Full Text Available Bacground/Aim. The therapy with intravesical instillation of bacillus Calmette-Guérin (BCG after transurethral resection (TUR of tumor is the gold standard of treatment of non-muscle invasive bladder cancer (NMIBC. The role and importance of BCG intravesical therapy in various shape of tumors, were confirmed by our previous investigation. The aim of this study was to examine whether incidence of recurrence and tumor regression differs depending on sex and age of patients. Methods. This study included a total of 899 patients suffering from NIMBC, treated at our institution from January 1, 2007 to March 1, 2013. Two groups of patients were formed: patients underwent TUR + BCG therapy (the group I and the group II with patients in whom TUR was performed as only therapy. These two groups of patients were divided into subgroups of respondents male and female, age 60 years or younger and older than 60 years. Statistical analysis was performed using χ2 test and the Kolmogorov-Smirnov test. Results. This research suggests that if the frequency of recurrence is seen as the only parameter, considering all the subjects, the lowest recurrence rate was determined in the male subjects, aged 60 years and younger who had received BCG after TUR. A high statistical significance was found in the incidence of recurrence in patients younger than 60 years, depending on the response to the therapy, while in those older than 60 years, the difference was at the level of statistical significance. This can be attributed to a certain degree of infravesical obstruction in older men. Conclusions. Sex and age of patients may have a significant influence on the course and outcome of NMIBC. The disease has the most malignant and most aggressive behavior when present in males older than 60 years.

  2. Do Standardised Prognostic Algorithms Reflect Local Practice? Application of EORTC Risk Tables for Non-Muscle Invasive (pTa/pT1 Bladder Cancer Recurrence and Progression in a Local Cohort

    Directory of Open Access Journals (Sweden)

    Rajiv Pillai

    2011-01-01

    Full Text Available A risk calculator algorithm to allow prediction of probabilities of 1- and 5-year recurrence and progression rates in individuals with pTa/pT1 bladder cancer has been proposed by the European Organisation for Research and Treatment of Cancer (EORTC and was incorporated into the European Association of Urology guidelines in 2006. We attempted to validate this algorithm in a cohort of patients with known outcome. Prognostic data were collected from a consecutively presenting cohort of 109 patients with non-muscle invasive (pTa/pT1 transitional cell cancer (TCC at a single institution between 1983 and 1985. Using the same statistical models as in the EORTC original paper, predicted probabilities of 1- and 5-year recurrence and progression were calculated. Patients were divided into four risk groups for recurrence (Ir-IVr and progression (Ip-IVp, respectively, using six prognostic criteria. These were then compared to the probabilities predicted in the EORTC algorithm. The predicted 1- and 5-year probabilities of recurrence were significantly higher in the study population as compared to the original EORTC algorithm for all four risk groups. The predicted 1-year probabilities for progression in groups Ip/IIIp and at 5-years for groups Ip/IIp were in accordance with the original algorithm, but were higher for the other progression groups. The concordance for the model of prediction using the study group for recurrence at 1 and 5 years was 62 and 63%, respectively, and for progression was 65 and 67, respectively. We were unable to validate the proposed algorithm in our group of patients. Although our study has limitations that prevent firm conclusions on the validity of the algorithm, it does expose some of the drawbacks of standardised nomograms when applied to local clinical practice.

  3. Structure and function of Drosophila unconventional myosin%果蝇非常规肌球蛋白的结构与功能

    Institute of Scientific and Technical Information of China (English)

    曹洋; 沈梅; 张洁; 李向东

    2011-01-01

    肌球蛋白是一类重要的分子马达,可以将ATP水解产生的能量转化成动能,沿由肌动蛋白组成的细丝运动.肌球蛋白构成一个大的基因家族,在许多细胞活动中起着重要作用,包括肌肉收缩、胞内转运、听觉、视觉等.果蝇基因组有13种肌球蛋白基因,包括2种常规肌球蛋白和11种非常规肌球蛋白.本文综述了近年来果蝇非常规肌球蛋白的研究进展.%Myosins are important molecular motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments.Myosins constitute a large superfamily and play key roles in a number of cellular processes including muscle contraction, intracellular trafficking, hearing, vision et al.Drosophila melanogaster has 13 myosin genes, including 2 conventional myosins and 11 unconventional myosins.In this review, we summarize recent progress in Drosophila unconventional myosins.

  4. Cardiac and skeletal muscle expression of mutant β-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy.

    Science.gov (United States)

    Di Domenico, Marina; Casadonte, Rita; Ricci, Pietroantonio; Santini, Mario; Frati, Giacomo; Rizzo, Antonietta; Carratelli, Caterina Romano; Lamberti, Monica; Parrotta, Elvira; Quaresima, Barbara; Faniello, Concetta M; Costanzo, Francesco; Cuda, Giovanni

    2012-10-01

    Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the β-myosin heavy chain (β-MHC) gene. In this study, the expression of the mutant β-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of β-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant β-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant β-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein.

  5. An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-Lin; ZHAO Jing; ZHANG Guan-Jun; KOU Jun-Ping; YU Bo-Yang

    2016-01-01

    Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity.The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method,using a known myosin Ⅱ ATPase inhibitor,blebbistatin.Several parameters were observed in the enzymatic reaction procedure,including the concentrations of the substrate (ATP) and calcium chloride,pH,and the reaction and incubation times.The proportion of coloration agent was also investigated.The sensitivity of this assay was compared with the malachite green method and bioluminescence method.Additionally,20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method.Our results showed that ATP at the concentration of 5 mmol·L-1 and ammonium molybdate:stannous chloride at the ratio of 15 ∶ 1 could greatly improve the sensitivity of this method.The IC50 of blebbistatin obtained by this method was consistent with literature.Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase.The optimized method showed similar accuracy,lower detecting limit,and wider linear range,which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.

  6. Intramanchette transport during primate spermiogenesis:expression of dynein, myosin Va, motor recruiter myosin Va,Ⅶa-Rab27a/b interacting protein, and Rab27b in the manchette during human and monkey spermiogenesis

    Institute of Scientific and Technical Information of China (English)

    Shinichi Hayasaka; Yukihiro Terada; Kichiya Suzuki; Haruo Murakawa; Ikuo Tachibana; Tadashi Sankai; Takashi Murakami; Nobuo Yaegashi; Kunihiro Okamura

    2008-01-01

    Aim: To show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, Ⅶa-Rab27a/b interacting protein (MyRIP), and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport (IMT). Methods: Spermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey (Macaca fascieularis). Immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the pro- teins were carried out. Samples were analyzed by light microscope. Results: Using RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis. Conclusion: We speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport. (Asian J Androl 2008 Jul; 10: 561-568)

  7. Switching of myosin-V motion between the lever-arm swing and brownian search-and-catch.

    Science.gov (United States)

    Fujita, Keisuke; Iwaki, Mitsuhiro; Iwane, Atsuko H; Marcucci, Lorenzo; Yanagida, Toshio

    2012-07-17

    Motor proteins are force-generating nanomachines that are highly adaptable to their ever-changing biological environments and have a high energy conversion efficiency. Here we constructed an imaging system that uses optical tweezers and a DNA handle to visualize elementary mechanical processes of a nanomachine under load. We apply our system to myosin-V, a well-known motor protein that takes 72 nm 'hand-over-hand' steps composed of a 'lever-arm swing' and a 'brownian search-and-catch'. We find that the lever-arm swing generates a large proportion of the force at low load (high load (1.9 pN), however, the contribution of the brownian search-and-catch increases to dominate, reaching 13 k(B)T of work. We believe the ability to switch between these two force-generation modes facilitates myosin-V function at high efficiency while operating in a dynamic intracellular environment.

  8. Shared gene structures and clusters of mutually exclusive spliced exons within the metazoan muscle myosin heavy chain genes.

    Directory of Open Access Journals (Sweden)

    Martin Kollmar

    Full Text Available Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs. The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis. Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both have independently been developed

  9. Thermal acclimation to cold alters myosin content and contractile properties of rainbow smelt, Osmerus mordax, red muscle.

    Science.gov (United States)

    Coughlin, David J; Shiels, Lisa P; Nuthakki, Seshuvardhan; Shuman, Jacie L

    2016-06-01

    Rainbow smelt (Osmerus mordax), a eurythermal fish, live in environments from -1.8 to 20°C, with some populations facing substantial annual variation in environmental temperature. These different temperature regimes pose distinct challenges to locomotion by smelt. Steady swimming performance, red muscle function and muscle myosin content were examined to assess the prediction that cold acclimation by smelt will lead to improved steady swimming performance and that any performance shift will be associated with changes in red muscle function and in its myosin heavy chain composition. Cold acclimated (4°C) smelt had a faster maximum steady swimming speed and swam with a higher tailbeat frequency than warm acclimated (10°C) smelt when tested at the same temperature (10°C). Muscle mechanics experiments demonstrated faster contractile properties in the cold acclimated fish when tested at 10°C. The red muscle of cold acclimated smelt had a shorter twitch times, a shorter relaxation times and a higher maximum shortening velocity. In addition, red muscle from cold acclimated fish displayed reduced thermal sensitivity to cold, maintaining higher force levels at 4°C compared to red muscle from warm acclimated fish. Immunohistochemistry suggests shifts in muscle myosin composition and a decrease in muscle cross-sectional area with cold acclimation. Dot blot analysis confirmed a shift in myosin content. Rainbow smelt do show a significant thermal acclimation response to cold. An examination of published values of maximum muscle shortening velocity in fishes suggests that smelt are particularly well suited to high levels of activity in very cold water. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The zebrafish goosepimples/myosin Vb mutant exhibits cellular attributes of human microvillus inclusion disease ☆ ☆☆

    OpenAIRE

    Sidhaye, Jaydeep; Pinto, Clyde Savio; Dharap, Shweta; Jacob, Tressa; Bhargava, Shobha; Sonawane, Mahendra

    2016-01-01

    Microvillus inclusion disease (MVID) is a life-threatening enteropathy characterised by malabsorption and incapacitating fluid loss due to chronic diarrhoea. Histological analysis has revealed that enterocytes in MVID patients exhibit reduction of microvilli, presence of microvillus inclusion bodies and intestinal villus atrophy, whereas genetic linkage analysis has identified mutations in myosin Vb gene as the main cause of MVID. In order to understand the cellular basis of MVID and the asso...

  11. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

    Science.gov (United States)

    Stathopoulou, Konstantina; Wittig, Ilka; Heidler, Juliana; Piasecki, Angelika; Richter, Florian; Diering, Simon; van der Velden, Jolanda; Buck, Friedrich; Donzelli, Sonia; Schröder, Ewald; Wijnker, Paul J M; Voigt, Niels; Dobrev, Dobromir; Sadayappan, Sakthivel; Eschenhagen, Thomas; Carrier, Lucie; Eaton, Philip; Cuello, Friederike

    2016-05-01

    Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure. © FASEB.

  12. Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish

    Directory of Open Access Journals (Sweden)

    Joshua Abrams

    2016-05-01

    Full Text Available Smooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11. Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt, which converts a highly conserved tryptophan to arginine (W512R in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y and coil-coiled (L1287M domains of Myh11 that fail to complement mlt. Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress.

  13. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    Science.gov (United States)

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  14. Interferon-γ causes cardiac myocyte atrophy via selective degradation of myosin heavy chain in a model of chronic myocarditis.

    Science.gov (United States)

    Cosper, Pippa F; Harvey, Pamela A; Leinwand, Leslie A

    2012-12-01

    Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    Science.gov (United States)

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  16. Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

    Directory of Open Access Journals (Sweden)

    James N. Cobley

    2016-04-01

    Full Text Available Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM. We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.

  17. Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi.

    Science.gov (United States)

    Chu, W Y; Chen, J; Zhou, R X; Zhao, F L; Meng, T; Chen, D X; Nong, X X; Liu, Z; Lu, S Q; Zhang, J S

    2011-04-01

    Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

  18. A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology.

    Science.gov (United States)

    Heredia, Dante J; Schubert, Douglas; Maligireddy, Siddhardha; Hennig, Grant W; Gould, Thomas W

    2016-01-01

    The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined

  19. A novel skeletal-myosin blocking drug for the study of neuromuscular physiology

    Directory of Open Access Journals (Sweden)

    Dante J Heredia

    2016-12-01

    Full Text Available The failure to transmit neural action potentials (APs into muscle APs is referred to as neuromuscular transmission failure (NTF. Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl-4-hydroxy-2H-chromen-2-one (BHC, previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as BTS, which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined in whole

  20. A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology

    Science.gov (United States)

    Heredia, Dante J.; Schubert, Douglas; Maligireddy, Siddhardha; Hennig, Grant W.; Gould, Thomas W.

    2016-01-01

    The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined

  1. Enhancement of force generated by individual myosin heads in skinned rabbit psoas muscle fibers at low ionic strength.

    Directory of Open Access Journals (Sweden)

    Haruo Sugi

    Full Text Available Although evidence has been presented that, at low ionic strength, myosin heads in relaxed skeletal muscle fibers form linkages with actin filaments, the effect of low ionic strength on contraction characteristics of Ca(2+-activated muscle fibers has not yet been studied in detail. To give information about the mechanism of muscle contraction, we have examined the effect of low ionic strength on the mechanical properties and the contraction characteristics of skinned rabbit psoas muscle fibers in both relaxed and maximally Ca(2+-activated states. By progressively decreasing KCl concentration from 125 mM to 0 mM (corresponding to a decrease in ionic strength μ from 170 mM to 50 mM, relaxed fibers showed changes in mechanical response to sinusoidal length changes and ramp stretches, which are consistent with the idea of actin-myosin linkage formation at low ionic strength. In maximally Ca(2+-activated fibers, on the other hand, the maximum isometric force increased about twofold by reducing KCl concentration from 125 to 0 mM. Unexpectedly, determination of the force-velocity curves indicated that, the maximum unloaded shortening velocity Vmax, remained unchanged at low ionic strength. This finding indicates that the actin-myosin linkages, which has been detected in relaxed fibers at low ionic strength, are broken quickly on Ca(2+ activation, so that the linkages in relaxed fibers no longer provide any internal resistance against fiber shortening. The force-velocity curves, obtained at various levels of steady Ca(2+-activated isometric force, were found to be identical if they are normalized with respect to the maximum isometric force. The MgATPase activity of muscle fibers during isometric force generation was found not to change appreciably at low ionic strength despite the two-fold increase in Ca(2+-activated isometric force. These results can be explained in terms of enhancement of force generated by individual myosin heads, but not by any

  2. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    Energy Technology Data Exchange (ETDEWEB)

    Lima, V.V. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil); Lobato, N.S.; Filgueira, F.P. [Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO (Brazil); Webb, R.C. [Department of Physiology, Georgia Regents University, Augusta, GA (United States); Tostes, R.C. [Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Giachini, F.R. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil)

    2014-08-15

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca{sup 2+}/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  3. The effect of the substitution of D{sub 2}O for H{sub 2}O on the degradation of myosin {beta} in solution by heat and by {sup 60}Co {gamma} radiation (1962); Effet de la substitution de D{sub 2}O a H{sub 2}O sur l'alteration de la Myosine B en solution par la chaleur et par les rayons {gamma} du {sup 60}CO (1962)

    Energy Technology Data Exchange (ETDEWEB)

    Pinset-Harstrom, I.; Fritsch, A. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1962-07-01

    (1) Alterations of myosin B produced by heat or irradiation are shown to be qualitatively identical as demonstrated by analytical centrifugation. (2) A considerable isotope effect was demonstrated using 75 per cent D{sub 2}O in the solvent. The sensitivity of myosin B to heat and irradiation is discussed in the light of this isotope effect. (3) Polymers appearing upon heat treatment of myosin B seem to be of a very different nature than the polymers occurring alter a similar treatment upon myosin A. Polymers obtained from myosin B can be depolymerized by ATP and they appear in a much narrower temperature range than myosin A polymers. This fact indicates a considerable difference in the activation enthalpies in the two reactions. (authors) [French] (1) Cette etude montre que les alterations de la myosine B provoquees par la chaleur et par l'irradiation aux rayons {gamma} sont - telles qu'elles apparaissent a l'ultracentrifugation analytique - qualitativement semblables. (2) Nous avons observe un effet isotopique considerable de la presence de 75 pour cent de D{sub 2}O dans le solvant sur la sensibilite de la myosine B envers ces deux agents, et nous avons presente une tentative d'explication de ce fait. (3) Les polymeres qui apparaissent apres un traitement par la chaleur de la myosine semblent etre d'une nature tres differente des polymeres que l'on voit apparaitre apres un traitement identique de la myosine A. Ceux obtenus a partir de le myosine B sont depolymerisables par l'intermediaire de l'ATP et apparaissent dans une zone de temperature beaucoup plus etroite que celles de la myosine A. Ce dernier fait indique une difference considerable de l'enthalpie d'activation des deux reactions. (auteurs)

  4. Myosin heavy chain composition of tiger (Panthera tigris) and cheetah (Acinonyx jubatus) hindlimb muscles.

    Science.gov (United States)

    Hyatt, Jon-Philippe K; Roy, Roland R; Rugg, Stuart; Talmadge, Robert J

    2010-01-01

    Felids have a wide range of locomotor activity patterns and maximal running speeds, including the very fast cheetah (Acinonyx jubatas), the roaming tiger (Panthera tigris), and the relatively sedentary domestic cat (Felis catus). As previous studies have suggested a relationship between the amount and type of activity and the myosin heavy chain (MHC) isoform composition of a muscle, we assessed the MHC isoform composition of selected hindlimb muscles from these three felid species with differing activity regimens. Using gel electrophoresis, western blotting, histochemistry, and immunohistochemistry with MHC isoform-specific antibodies, we compared the MHC composition in the tibialis anterior, medial gastrocnemius (MG), plantaris (Plt), and soleus muscles of the tiger, cheetah, and domestic cat. The soleus muscle was absent in the cheetah. At least one slow (type I) and three fast (types IIa, IIx, and IIb) MHC isoforms were present in the muscles of each felid. The tiger had a high combined percentage of the characteristically slower isoforms (MHCs I and IIa) in the MG (62%) and the Plt (86%), whereas these percentages were relatively low in the MG (44%) and Plt (55%) of the cheetah. In general, the MHC isoform characteristics of the hindlimb muscles matched the daily activity patterns of these felids: the tiger has daily demands for covering long distances, whereas the cheetah has requirements for speed and power. (c) 2009 Wiley-Liss, Inc.

  5. Myosin Binding Protein-C Slow: An Intricate Subfamily of Proteins

    Directory of Open Access Journals (Sweden)

    Maegen A. Ackermann

    2010-01-01

    Full Text Available Myosin binding protein C (MyBP-C consists of a family of thick filament associated proteins. Three isoforms of MyBP-C exist in striated muscles: cardiac, slow skeletal, and fast skeletal. To date, most studies have focused on the cardiac form, due to its direct involvement in the development of hypertrophic cardiomyopathy. Here we focus on the slow skeletal form, discuss past and current literature, and present evidence to support that: (i MyBP-C slow comprises a subfamily of four proteins, resulting from complex alternative shuffling of the single MyBP-C slow gene, (ii the four MyBP-C slow isoforms are expressed in variable amounts in different skeletal muscles, (iii at least one MyBP-C slow isoform is preferentially found at the periphery of M-bands and (iv the MyBP-C slow subfamily may play important roles in the assembly and stabilization of sarcomeric M- and A-bands and regulate the contractile properties of the actomyosin filaments.

  6. Cofilin Regulates Nuclear Architecture through a Myosin-II Dependent Mechanotransduction Module

    Science.gov (United States)

    Wiggan, O’Neil; Schroder, Bryce; Krapf, Diego; Bamburg, James R.; DeLuca, Jennifer G.

    2017-01-01

    Structural features of the nucleus including shape, size and deformability impact its function affecting normal cellular processes such as cell differentiation and pathological conditions such as tumor cell migration. Despite the fact that abnormal nuclear morphology has long been a defining characteristic for diseases such as cancer relatively little is known about the mechanisms that control normal nuclear architecture. Mounting evidence suggests close coupling between F-actin cytoskeletal organization and nuclear morphology however, mechanisms regulating this coupling are lacking. Here we identify that Cofilin/ADF-family F-actin remodeling proteins are essential for normal nuclear structure in different cell types. siRNA mediated silencing of Cofilin/ADF provokes striking nuclear defects including aberrant shapes, nuclear lamina disruption and reductions to peripheral heterochromatin. We provide evidence that these anomalies are primarily due to Rho kinase (ROCK) controlled excessive contractile myosin-II activity and not to elevated F-actin polymerization. Furthermore, we demonstrate a requirement for nuclear envelope LINC (linker of nucleoskeleton and cytoskeleton) complex proteins together with lamin A/C for nuclear aberrations induced by Cofilin/ADF loss. Our study elucidates a pivotal regulatory mechanism responsible for normal nuclear structure and which is expected to fundamentally influence nuclear function. PMID:28102353

  7. Myosin phosphorylation and force potentiation in skeletal muscle: evidence from animal models.

    Science.gov (United States)

    Vandenboom, Rene; Gittings, William; Smith, Ian C; Grange, Robert W; Stull, James T

    2013-12-01

    The contractile performance of mammalian fast twitch skeletal muscle is history dependent. The effect of previous or ongoing contractile activity to potentiate force, i.e. increase isometric twitch force, is a fundamental property of fast skeletal muscle. The precise manifestation of force potentiation is dependent upon a variety of factors with two general types being identified; staircase potentiation referring to the progressive increase in isometric twitch force observed during low frequency stimulation while posttetanic potentiation refers to the step-like increase in isometric twitch force observed following a brief higher frequency (i.e. tetanic) stimulation. Classic studies established that the magnitude and duration of potentiation depends on a number of factors including muscle fiber type, species, temperature, sarcomere length and stimulation paradigm. In addition to isometric twitch force, more recent work has shown that potentiation also influences dynamic (i.e. concentric and/or isotonic) force, work and power at a range of stimulus frequencies in situ or in vitro, an effect that may translate to enhanced physiological function in vivo. Early studies performed on both intact and permeabilized models established that the primary mechanism for this modulation of performance was phosphorylation of myosin, a modification that increased the Ca(2+) sensitivity of contraction. More recent work from a variety of muscle models indicates, however, the presence of a secondary mechanism for potentiation that may involve altered Ca(2+) handling. The primary purpose of this review is to highlight these recent findings relative to the physiological utility of force potentiation in vivo.

  8. Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

    Science.gov (United States)

    Hegan, Peter S; Ostertag, Eric; Geurts, Aron M; Mooseker, Mark S

    2015-10-01

    In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.

  9. Mutant p53-associated myosin-X upregulation promotes breast cancer invasion and metastasis.

    Science.gov (United States)

    Arjonen, Antti; Kaukonen, Riina; Mattila, Elina; Rouhi, Pegah; Högnäs, Gunilla; Sihto, Harri; Miller, Bryan W; Morton, Jennifer P; Bucher, Elmar; Taimen, Pekka; Virtakoivu, Reetta; Cao, Yihai; Sansom, Owen J; Joensuu, Heikki; Ivaska, Johanna

    2014-03-01

    Mutations of the tumor suppressor TP53 are present in many forms of human cancer and are associated with increased tumor cell invasion and metastasis. Several mechanisms have been identified for promoting dissemination of cancer cells with TP53 mutations, including increased targeting of integrins to the plasma membrane. Here, we demonstrate a role for the filopodia-inducing motor protein Myosin-X (Myo10) in mutant p53-driven cancer invasion. Analysis of gene expression profiles from 2 breast cancer data sets revealed that MYO10 was highly expressed in aggressive cancer subtypes. Myo10 was required for breast cancer cell invasion and dissemination in multiple cancer cell lines and murine models of cancer metastasis. Evaluation of a Myo10 mutant without the integrin-binding domain revealed that the ability of Myo10 to transport β₁ integrins to the filopodia tip is required for invasion. Introduction of mutant p53 promoted Myo10 expression in cancer cells and pancreatic ductal adenocarcinoma in mice, whereas suppression of endogenous mutant p53 attenuated Myo10 levels and cell invasion. In clinical breast carcinomas, Myo10 was predominantly expressed at the invasive edges and correlated with the presence of TP53 mutations and poor prognosis. These data indicate that Myo10 upregulation in mutant p53-driven cancers is necessary for invasion and that plasma-membrane protrusions, such as filopodia, may serve as specialized metastatic engines.

  10. Contractile properties and myosin expression in rats born and reared in hypergravity.

    Science.gov (United States)

    Picquet, F; Bouet, V; Canu, M H; Stevens, L; Mounier, Y; Lacour, M; Falempin, M

    2002-06-01

    The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.

  11. Segmental distribution of myosin heavy chain isoforms within single muscle fibers.

    Science.gov (United States)

    Zhang, Ming; Gould, Maree

    2017-02-18

    Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the 3 EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I&cenveo_unknown_entity_wingdings_F0F3;IIA&cenveo_unknown_entity_wingdings_F0F3;IIX&cenveo_unknown_entity_wingdings_F0F3;IIB, however in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be due to the myonuclear domain. This article is protected by copyright. All rights reserved.

  12. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    Science.gov (United States)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  13. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    Science.gov (United States)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  14. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology.

    Science.gov (United States)

    Carrier, Lucie; Mearini, Giulia; Stathopoulou, Konstantina; Cuello, Friederike

    2015-12-01

    More than 350 individual MYPBC3 mutations have been identified in patients with inherited hypertrophic cardiomyopathy (HCM), thus representing 40–50% of all HCM mutations, making it the most frequently mutated gene in HCM. HCM is considered a disease of the sarcomere and is characterized by left ventricular hypertrophy, myocyte disarray and diastolic dysfunction. MYBPC3 encodes for the thick filament associated protein cardiac myosin-binding protein C (cMyBP-C), a signaling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of contraction and relaxation. This review aims to provide a succinct overview of how mutations in MYBPC3 are considered to affect the physiological function of cMyBP-C, thus causing the deleterious consequences observed inHCM patients. Importantly, recent advances to causally treat HCM by repairing MYBPC3 mutations by gene therapy are discussed here, providing a promising alternative to heart transplantation for patients with a fatal form of neonatal cardiomyopathy due to bi-allelic truncating MYBPC3 mutations.

  15. Effect of myosin heavy chain composition of muscles on meat quality in Laiwu pigs and Duroc

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to explain the mechanism of high meat quality in Laiwu pigs and investigate the relation between myosin heavy chains (MyHC) composition and meat quality, meat quality analysis was conducted and mRNA expression of MyHC I, IIa, IIx, IIb was quantified by real-time fluorescence PCR in longissimus muscle (LM) and semimembranous muscle of Laiwu pigs and Duroc. The result indicated that, compared with Duroc, mRNA expression of MyHC IIa, IIx in LM and semimembranous muscle of Laiwu pigs was significantly increased, mRNA expression of MyHC IIb was dramatically decreased. However, the expression of MyHC I was not significantly affected by breeds. The correlation between mRNA expression of MyHC I, IIa, IIx in LM and meat color, pH value, marbling, intramuscular fat content was positive, but shear value of LM was negative. The relation between MyHC IIb mRNA expression and marbling, intramuscular fat content was dramatically negative, whereas shear value was strikingly positive, as well as fiber diameter, but without reaching statistical significance. Therefore, the composition of MyHC I, IIa, IIx, IIb affected meat quality, furthermore, expression of MyHC I, IIa, IIx, IIb mRNA prominently influenced meat characteristics, especially edible quality of muscle, suggesting that mRNA expression level of MyHC I, IIa, IIx, IIb can exactly and impersonally estimate meat quality.

  16. Effect of myosin heavy chain composition of muscles on meat quality in Laiwu pigs and Duroc

    Institute of Scientific and Technical Information of China (English)

    HU HongMei; WANG JiYing; ZHU RongSheng; GUO JianFeng; WU Ying

    2008-01-01

    In order to explain the mechanism of high meat quality in Laiwu pigs and investigate the relation between myosin heavy chains (MyHC) composition and meat quality, meat quality analysis was conducted and mRNA expression of MyHC I, IIa, IIx, IIb was quantified by real-time fluorescence PCR in Iongissimus muscle (LM) and semimembranous muscle of Laiwu pigs and Duroc. The result indicated that, compared with Duroc, mRNA expression of MyHC IIa, IIx in LM and semimembranous muscle of Laiwu pigs was significantly increased, mRNA expression of MyHC Ilia'was dramatically decreased.However, the expression of MyHC I was not significantly affected by breeds. The correlation between mRNA expression of MyHC I, IIa, IIx in LM and meat color, pH value, marbling, intramuscular fat content was positive, but shear value of LM was negative. The relation between MyHC lib mRNA expression and marbling, intramuscular fat content was dramatically negative, whereas shear value was strikingly positive, as well as fiber diameter, but without reaching statistical significance. Therefore, the composition of MyHC I, IIa, IIx, IIb affected meat quality, furthermore, expression of MyHC I,IIa, IIx, IIb mRNA prominently influenced meat characteristics, especially edible quality of muscle, suggesting that mRNA expression level of MyHC I, IIa, IIx, IIb can exactly and impersonally estimate meat quality.

  17. Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin-myosin Va interaction.

    Science.gov (United States)

    Chang, Huikyoung; Choi, Hyunjung; Joo, Kyung-Mi; Kim, Daegun; Lee, Tae Ryong

    2012-11-01

    Human skin hyperpigmentation disorders occur when the synthesis and/or distribution of melanin increases. The distribution of melanin in the skin is achieved by melanosome transport and transfer. The transport of melanosomes, the organelles where melanin is made, in a melanocyte precedes the transfer of the melanosomes to a keratinocyte. Therefore, hyperpigmentation can be regulated by decreasing melanosome transport. In this study, we found that an extract of Saururus chinensis Baill (ESCB) and one of its components, manassantin B, inhibited melanosome transport in Melan-a melanocytes and normal human melanocytes (NHMs). Manassantin B disturbed melanosome transport by disrupting the interaction between melanophilin and myosin Va. Manassantin B is neither a direct nor an indirect inhibitor of tyrosinase. The total melanin content was not reduced when melanosome transport was inhibited in a Melan-a melanocyte monoculture by manassantin B. Manassantin B decreased melanin content only when Melan-a melanocytes were co-cultured with SP-1 keratinocytes or stimulated by α-MSH. Therefore, we propose that specific inhibitors of melanosome transport, such as manassantin B, are potential candidate or lead compounds for the development of agents to treat undesirable hyperpigmentation of the skin.

  18. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

    Science.gov (United States)

    Cao, Ruihua; Huang, Fei; Wang, Peihuan; Chen, Chen; Zhu, Guoxiong; Chen, Lei; Wu, Gaoyi

    2015-05-01

    To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ.

  19. An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus.

    Science.gov (United States)

    Nguyen, L T; Stephenson, G M

    1999-10-01

    In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

  20. Dose-dependent electrophysiological effects of the myosin activator omecamtiv mecarbil in canine ventricular cardiomyocytes.

    Science.gov (United States)

    Szentandrassy, N; Horvath, B; Vaczi, K; Kistamas, K; Masuda, L; Magyar, J; Banyasz, T; Papp, Z; Nanasi, P P

    2016-08-01

    Omecamtiv mecarbil (OM) is a myosin activator agent recently developed for treatment of heart failure. Although its action on extending systolic ejection time and increasing left ventricular ejection fraction is well documented, no data is available regarding its possible side-effects on cardiac ion channels. Therefore, the present study was designed to investigate the effects of OM on action potential morphology and the underlying ion currents in isolated canine ventricular myocytes using sharp microelectrodes, conventional patch clamp, and action potential voltage clamp techniques. OM displayed a concentration-dependent action on action potential configuration: 1 μM OM had no effect, while action potential duration and phase-1 repolarization were reduced and the plateau potential was depressed progressively at higher concentrations (10 - 100 μM; P < 0.05 compared to control). Accordingly, OM (10 μM) decreased the density of the transient outward K(+) current (Ito), the L-type Ca(2+) current (ICa) and the rapid delayed rectifier K(+) current (IKr), but failed to modify the inward rectifier K(+) current (IK1). It is concluded, that although the therapeutic concentrations of OM are not likely to influence cardiac ion currents significantly, alterations of the major cardiac ion currents can be anticipated at concentrations above those clinically tolerated.

  1. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    Science.gov (United States)

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  2. Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses.

    Science.gov (United States)

    Roux, Isabelle; Hosie, Suzanne; Johnson, Stuart L; Bahloul, Amel; Cayet, Nadège; Nouaille, Sylvie; Kros, Corné J; Petit, Christine; Safieddine, Saaid

    2009-12-01

    The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

  3. Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation.

    Science.gov (United States)

    Tiago, Teresa; Ramos, Susana; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2006-03-31

    Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.

  4. Helicobacter pylori CagA disrupts epithelial patterning by activating myosin light chain.

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    Jonathan B Muyskens

    Full Text Available Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC, a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagA(EPISA induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagA(EPISA and CagA have equivalent subcellular localization, CagA(EPISA is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity.

  5. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    Science.gov (United States)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  6. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    Science.gov (United States)

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  7. The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

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    David A Tumbarello

    2015-10-01

    Full Text Available Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.

  8. A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

    Science.gov (United States)

    Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

    2014-01-01

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

  9. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

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    Toshiyuki Mizui

    Full Text Available The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1 chemical long-term potentiation (LTP stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2 Ca(2+ influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3 the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK or Rho-associated kinase (ROCK inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  10. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

    Science.gov (United States)

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  11. Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement.

    Science.gov (United States)

    Lombardi, Maria L; Knecht, David A; Dembo, Micah; Lee, Juliet

    2007-05-01

    Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA-) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE-) and mhcA- cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

  12. Regenerating tail muscles in lizard contain Fast but not Slow Myosin indicating that most myofibers belong to the fast twitch type for rapid contraction.

    Science.gov (United States)

    Alibardi, L

    2015-10-01

    During tail regeneration in lizards a large mass of muscle tissue is formed in form of segmental myomeres of similar size located under the dermis of the new tail. These muscles accumulate glycogen and a fast form of myosin typical for twitch myofibers as it is shown by light and ultrastructural immunocytochemistry using an antibody directed against a Fast Myosin Heavy Chain. High resolution immunogold labeling shows that an intense labeling for fast myosin is localized over the thick filaments of the numerous myofibrils in about 70% of the regenerated myofibers while the labeling becomes less intense in the remaining muscle fibers. The present observations indicate that at least two subtypes of Fast Myosin containing muscle fibers are regenerated, the prevalent type was of the fast twitch containing few mitochondria, sparse glycogen, numerous smooth endoplasmic reticulum vesicles. The second, and less frequent type was a Fast-Oxidative-Glycolitic twitch fiber containing more mitochondria, a denser cytoplasm and myofibrils. Since their initial differentiation, myoblasts, myotubes and especially the regenerated myofibers do not accumulate any immuno-detectable Slow Myosin Heavy Chain. The study indicates that most of the segmental muscles of the regenerated tail serve for the limited bending of the tail during locomotion and trashing after amputation of the regenerated tail, a phenomenon that facilitates predator escape.

  13. Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle.

    Science.gov (United States)

    Perry, Christopher G R; Kane, Daniel A; Lin, Chien-Te; Kozy, Rachel; Cathey, Brook L; Lark, Daniel S; Kane, Constance L; Brophy, Patricia M; Gavin, Timothy P; Anderson, Ethan J; Neufer, P Darrell

    2011-07-15

    Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

  14. Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles.

    Science.gov (United States)

    Ali, M Yusuf; Vilfan, Andrej; Trybus, Kathleen M; Warshaw, David M

    2016-11-15

    Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors-which in turn slows down the leading motor-and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

  15. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

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    Nasibeh Yousefzadeh

    2016-01-01

    Full Text Available Abstract Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05 and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05 were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201% and α- MHC expression was lower (47% than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart.

  16. Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure.

    Science.gov (United States)

    Neary, Marianne T; Neary, Joseph M; Lund, Gretchen K; Holt, Timothy N; Garry, Franklyn B; Mohun, Timothy J; Breckenridge, Ross A

    2014-09-01

    Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension.

  17. Myosin light chain kinase mediates intestinal barrier disruption following burn injury.

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    Chuanli Chen

    Full Text Available BACKGROUND: Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC phosphorylation mediated by MLC kinase (MLCK is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. METHODOLOGY/PRINCIPAL FINDINGS: Male balb/c mice were assigned randomly to either sham burn (control or 30% total body surface area (TBSA full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg, an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. CONCLUSIONS/SIGNIFICANCE: The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.

  18. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

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    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta (IIT); (Iowa); (Miami-MED)

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  19. Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

    Directory of Open Access Journals (Sweden)

    Orion D Weiner

    2006-02-01

    Full Text Available Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes, scaffolded by hematopoietic protein 1 (Hem-1, that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b substantially weakens Rac activation and phosphatidylinositol-(3,4,5-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5-tris-phosphate/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

  20. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

  1. Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity.

    Science.gov (United States)

    Eddinger, T J; Meer, D P

    2001-02-01

    Isolated single smooth muscle cells (SMCs) from different regions of the rabbit stomach were used to determine a possible correlation between unloaded shortening velocity and smooth muscle (SM) myosin heavy chain (MHC) S1 head isoform composition (SMA, no head insert; SMB, with head insert). alpha-Toxin-permeabilized isolated single cells were maximally activated to measure unloaded shortening velocity and subsequently used in an RT-PCR reaction to determine the SMA/SMB content of the same cell. SM MHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 +/- 7.3% SMB; n = 16); cells from the antrum express primarily SMB (94.9 +/- 1.0% SMB; n = 16). Mean fundic cell unloaded shortening velocity was 0.014 +/- 0.002 cell lengths/s compared with 0.036 +/- 0.002 for the antrum cells. Unloaded shortening velocity in these cells was significantly correlated with their percent SMB expression (r2 = 0.58). Resting cell length does not correlate with the percent SMB expression (n = 32 cells). Previously published assays of purified or expressed SMA and SMB heavy meromyosin show a twofold difference in actin filament sliding speed in in vitro motility assays. Extrapolation of our data to 0-100% SMB would give a 10-fold range of shortening velocity, which is closer to the approximately 20-fold range reported from various SM tissues. This suggests that mechanisms in addition to the MHC S1 head isoforms regulate shortening velocity.

  2. Inhibiting Myosin-ATPase Reveals Dynamic Range of Mitochondrial Respiratory Control in Skeletal Muscle

    Science.gov (United States)

    Perry, Christopher G.R.; Kane, Daniel A.; Lin, Chien-Te; Kozy, Rachel; Cathey, Brook L.; Lark, Daniel S.; Kane, Constance L.; Brophy, Patricia M.; Gavin, Timothy P; Anderson, Ethan J.; Neufer, P. Darrell

    2013-01-01

    Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (∼20-300 μM) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >∼250 and ∼80 μM in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ∼2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30°C but not 37°C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 μM) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ∼1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state. PMID:21554250

  3. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

    Science.gov (United States)

    Yousefzadeh, Nasibeh; Jeddi, Sajad; Alipour, Mohammad Reza

    2016-01-01

    Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 bea