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Sample records for acetylating myosin light

  1. 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

  2. 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.

  3. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    Science.gov (United States)

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  4. 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.

  5. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    Science.gov (United States)

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style. PMID:19648394

  6. Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

    OpenAIRE

    Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Steven D Lidofsky

    2011-01-01

     The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin ...

  7. THE CHARACTERIZATION OF MYOSIN LIGHT CHAIN PHOSPHORYLATION BY THE CONSTITUTIVELY ACTIVE FRAGMENT OF MLCK

    Institute of Scientific and Technical Information of China (English)

    杨静娴; 王晓明; 唐泽耀; 陈华; 戴淑芳; 林原

    2003-01-01

    Objective. To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF) on Ca2+-CaM dependent phosphorylation of myosin light chains (CDPM) of smooth muscle. Methods. Proteolysis of myosin light chain kinase (MLCK) by trypsin was used to obtain the constitutively active fragment of MLCK; Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK. The phosphorylation of myosin light chains was detected using SDS-PAGE and Scoin Image Software, and myosin Mg2+-ATPase activities were measured using spectrophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimulating myosin Mg2+-ATPase activities compared with intact MLCK. Results. Our assay demonstrated that MLCKF had a specific activity on CDPM. We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations, different incubation time and different incubation temperatures. CDPM by MLCKF was also less influenced by the change of ionic strength of KC1 than CDPM by intact MLCK. Mg2+--ATPase activities of phosphorylat- ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and different reaction time. These differences were statistically significant (**P<0.01, *P<0.05). Conclusion. MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg2*-ATPase activities. Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.

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

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    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.

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

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    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.

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

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    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.

  11. Myosin light chain kinase-dependent microvascular hyperpermeability in thermal injury.

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    Huang, Qiaobing; Xu, Wenjuan; Ustinova, Elena; Wu, Mack; Childs, Ed; Hunter, Felicia; Yuan, Sarah

    2003-10-01

    Although the critical role of systemic inflammatory edema in the development of multiple organ failure in patients with massive burns has been fully recognized, the precise mechanisms responsible for the accumulation of blood fluid and proteins in tissues remote from the burn wound are poorly understood. The aim of this study was to test the hypothesis that circulating factors released during thermal injury cause microvascular leakage by triggering endothelial cell contraction and barrier dysfunction. A third-degree scald burn was induced in rats on the dorsal skin covering 25% total body surface area. The microcirculation and transvascular flux of albumin were observed in the rat mesentery using intravital fluorescence microscopy. The direct effect of circulating factors on microvascular barrier function was assessed by measuring the apparent permeability coefficient of albumin in isolated rat mesenteric venules during perfusion of plasma freshly withdrawn from burned rats. The in vivo study showed that the transvenular flux of albumin was significantly increased over a 6-h period with a maximal response seen at 3 h postburn. Importantly, perfusion of noninjured venules with burn plasma induced a time-dependent increase in albumin permeability. Pharmacological inhibition of protein kinase C, Src tyrosine kinases, or mast cell activation did not significantly affect the hyperpermeability response; however, blockage of myosin light chain phosphorylation with the myosin light chain kinase inhibitor ML-7 greatly attenuated the burn-induced increase in venular permeability in a dose-related pattern. The results support a role for endogenous circulating factors in microvascular leakage during burns. Myosin light chain phosphorylation-dependent endothelial contractile response may serve as an end-point effector leading to microvascular barrier dysfunction. PMID:14501951

  12. Dynamic light scattering study of the effect of Mg2+ and ATP on synthetic myosin filaments.

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    Takayama, S.; Fujime, S

    1995-01-01

    The dynamic light scattering (DLS) method provides us with information about the apparent diffusion coefficient, Dapp, as well as the static scattering intensity, Is, of particles in solution. For long but thin rods with length L and diameter d, the dependence on L and d of Dapp is quite different from that of Is. By means of DLS we studied synthetic myosin filaments of rabbit skeletal muscle in solution at pH 8.3 and 10 degrees C. It appeared that Mg2+ ions induced thickening and lengthening...

  13. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

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    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  14. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

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    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz

    2012-01-01

    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  15. A Novel Myosin Essential Light Chain Mutation Causes Hypertrophic Cardiomyopathy with Late Onset and Low Expressivity

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    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.

  16. 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.

  17. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

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    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  18. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

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    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.

  19. 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. PMID:27483374

  20. 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.

  1. Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

    OpenAIRE

    Zahs, Anita; Bird, Melanie D.; Ramirez, Luis; Turner, Jerrold R; Choudhry, Mashkoor A.; Kovacs, Elizabeth J

    2012-01-01

    Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory...

  2. 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. PMID:27162358

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. 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.

  10. 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.

  11. 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.

  12. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    Science.gov (United States)

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  13. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    OpenAIRE

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2009-01-01

    To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC pro...

  14. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    Directory of Open Access Journals (Sweden)

    Fabian Lacramioara

    2007-05-01

    Full Text Available Abstract Background Phosphatidylinositol 4,5-bisphosphate (PIP2 is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG and inositol trisphosphate (IP3, which in turn induces calcium (Ca2+ release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM. In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.

  15. 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.

  16. CLONING AND CHARACTERISATION OF ALKALI MYOSIN LIGHT CHAIN GENE (MLC-3 OF CATTLE FILARIAL PARASITE SETARIA DIGITATA

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    Arumugam Murugananthan, Eric Hamilton Karunanayake*, Kamani Hemamala Tennekoon

    2010-11-01

    Full Text Available Lymphatic filariasis is a tropical disease caused by filarial parasites including Wuchereria bancrofti. Although bancroftian filariasis causes severe disabling and debilitating clinical conditions in human, very little is known about the molecular biology of the parasite. The paucity of parasitic material is the main reason for this lack of knowledge. Setaria digitata is a cattle filarial parasite, closely resembling W. bancrofti in many aspects. Therefore it can be used as a model organism to study W. bancrofti. In the present study, the genomic library of S. digitata adult parasites was constructed and probed with a 32P labeled partial mRNA sequence PCR amplified from a previously isolated cDNA clone containing a 661 bp mRNA transcript of S. digitata alkali myosin light chain gene. Isolated positive clones were sequenced and edited by using bioinformatics tools. Though the 5´ flanking region did not reveal any consensus TATA box sequences, a potential CAAT box like sequence, CCAAT and seven possible transcription factor elements were identified. The entire gene had four exons encoding 149 amino acids interrupted by three introns of varying lengths of 87, 295 and 69 bp respectively. Sequences around the splice junctions were fairly conserved and agreed with the general GT-AG splicing rule. The 3´ flanking region consists of three putative polyadenylation signals with the sequence AATAAA. The gene was AT rich with a GC content of 35%. Southern hybridisation studies suggested that this gene is likely to be a single-copy gene. Homology search of amino acid sequences showed more than 80% similarity with Caenorhabditis species and 40-50% with other vertebrate and invertebrate myosin light chains. Analysis of the amino acid sequence with the NCBI conserved domain database for interactive domain family identified the protein as a member of calcium binding protein family as it comprised of two highly conserved EF hand motifs, and may suggest a

  17. Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

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    Shelly Woody

    Full Text Available Myosin II (MyoII is required for insulin-responsive glucose transporter 4 (GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC of MyoIIA via myosin light chain kinase (MLCK. The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy ethane-N,N,N',N'-tetra acetic acid, (BAPTA (in the presence of insulin impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

  18. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

    Science.gov (United States)

    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  19. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    Energy Technology Data Exchange (ETDEWEB)

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.; (IIT); (Vermont); (Duke)

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  20. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    OpenAIRE

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

    2008-01-01

    Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that my...

  1. Gene expression patterns in transgenic mouse models of hypertrophic cardiomyopathy caused by mutations in myosin regulatory light chain.

    Science.gov (United States)

    Huang, Wenrui; Kazmierczak, Katarzyna; Zhou, Zhiqun; Aguiar-Pulido, Vanessa; Narasimhan, Giri; Szczesna-Cordary, Danuta

    2016-07-01

    Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58→Glutamine (R58Q) and Aspartic Acid 166 → Valine (D166V), and one benign, Lysine 104 → Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms. PMID:26906074

  2. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  3. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    Science.gov (United States)

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  4. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    Science.gov (United States)

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

  5. Remote control of myosin and kinesin motors using light-activated gearshifting.

    Science.gov (United States)

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C; Schindler, Tony D; Nogales, Eva; Bryant, Zev

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

  6. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    OpenAIRE

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2007-01-01

    The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is ...

  7. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    Science.gov (United States)

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  8. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    Science.gov (United States)

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (pmutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (pthrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  9. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    Science.gov (United States)

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene. PMID:25911560

  10. Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

    Directory of Open Access Journals (Sweden)

    Kenji Takemoto

    Full Text Available Mechanical stress that arises due to deformation of the extracellular matrix (ECM either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC via the RhoA/RhoA-associated protein kinase (ROCK pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853. Because myosin phosphatase targeting subunit 1 (Thr853 is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188 that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188 induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188. Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

  11. Phosphorylation and the N-terminal Extension of the Regulatory Light Chain Help Orient and Align the Myosin Heads in Drosophila Flight Muscle

    OpenAIRE

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2009-01-01

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2Δ2-46) or disruption of the phosphorylation sites by substituting alanines (Dmlc2S66A, S67A) decreased the equatorial intensity ratio (I20/I10), indicating decreased myosin mass associated with the thin f...

  12. The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscle.

    OpenAIRE

    Moore, J R; Dickinson, M H; Vigoreaux, J O; Maughan, D W

    2000-01-01

    The Drosophila myosin regulatory light chain (DMLC2) is homologous to MLC2s of vertebrate organisms, except for the presence of a unique 46-amino acid N-terminal extension. To study the role of the DMLC2 N-terminal extension in Drosophila flight muscle, we constructed a truncated form of the Dmlc2 gene lacking amino acids 2-46 (Dmlc2(Delta2-46)). The mutant gene was expressed in vivo, with no wild-type Dmlc2 gene expression, via P-element-mediated germline transformation. Expression of the tr...

  13. Light-dark regulation of sulfate assimilation in Lemna minor L. in the presence of o-acetyl-l-serine

    International Nuclear Information System (INIS)

    The effect of light removal and addition of O-acetyl-l-serine (OAS) on sulfate assimilation in Lemna minor L. was analyzed by measuring the extractable activity of adenosine 5'-phosphosulfate sulfotransferase (APSSTase) and the in vivo incorporation of 35SO42-. After removal of light APSSTase activity decreased to 10% within 24 h in the absence and to 50% in the presence of OAS. Within 24 h total 35SO42- uptake decreased to 60% without and increased to 130% with OAS compared to light controls. The incorporation of 35S into cysteine increased 2 times without and 15 times with OAS, labelling of glutathione decreased to 65% and increased to 140%, the one of the protein fraction decreased to 30% and to 20% of the light control in the absence and presence of OAS. Our results indicate that OAS has a regulatory function on the assimilation of sulfate and that protein synthesis is inhibited in the dark

  14. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ying [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China); Li, Jianguo, E-mail: 2010lijianguo@sina.cn [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  15. Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

    Science.gov (United States)

    Jalagadugula, Gauthami; Mao, Guangfen; Kaur, Gurpreet; Goldfinger, Lawrence E.; Dhanasekaran, Danny N.

    2010-01-01

    Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region −729/−542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations. PMID:20876458

  16. Neuregulin1–β decreases interleukin–1β–induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability

    Science.gov (United States)

    Wu, Limin; Ramirez, Servio H.; Andrews, Allison M.; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H.; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. PMID:26438054

  17. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Science.gov (United States)

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  18. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    Directory of Open Access Journals (Sweden)

    Rupalatha Maddala

    Full Text Available Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations

  19. Protein multi-scale organization through graph partitioning and robustness analysis: application to the myosin–myosin light chain interaction

    International Nuclear Information System (INIS)

    Despite the recognized importance of the multi-scale spatio-temporal organization of proteins, most computational tools can only access a limited spectrum of time and spatial scales, thereby ignoring the effects on protein behavior of the intricate coupling between the different scales. Starting from a physico-chemical atomistic network of interactions that encodes the structure of the protein, we introduce a methodology based on multi-scale graph partitioning that can uncover partitions and levels of organization of proteins that span the whole range of scales, revealing biological features occurring at different levels of organization and tracking their effect across scales. Additionally, we introduce a measure of robustness to quantify the relevance of the partitions through the generation of biochemically-motivated surrogate random graph models. We apply the method to four distinct conformations of myosin tail interacting protein, a protein from the molecular motor of the malaria parasite, and study properties that have been experimentally addressed such as the closing mechanism, the presence of conserved clusters, and the identification through computational mutational analysis of key residues for binding

  20. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

    Directory of Open Access Journals (Sweden)

    Cian M McCrudden

    Full Text Available Therapeutic inhibition of poly(ADP-ribose polymerase (PARP, as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699, 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 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

  1. 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.

  2. All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

    Science.gov (United States)

    Zuo, Li; Yang, Xiaoping; Lu, Man; Hu, Ruolei; Zhu, Huaqing; Zhang, Sumei; Zhou, Qing; Chen, Feihu; Gui, Shuyu; Wang, Yuan

    2016-10-01

    All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway. PMID:27564600

  3. Characterizations of myosin essential light chain’s N-terminal truncation mutant Δ43 in transgenic mouse papillary muscles by using tension transients in response to sinusoidal length alterations

    OpenAIRE

    Wang, Li; Muthu, Priya; Szczesna-Cordary, Danuta; Kawai, Masataka

    2013-01-01

    Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants w...

  4. Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction via tight junction regulation in a rabbit model of atherosclerosis.

    Science.gov (United States)

    Cheng, Xiaowen; Wang, Xiaobian; Wan, Yufeng; Zhou, Qing; Zhu, Huaqing; Wang, Yuan

    2015-09-01

    Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet‑induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)‑1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high‑fat diet‑fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high‑fat diet (AS group) or a high‑fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium‑dependent relaxation and endothelium‑independent relaxation were measured using high-frequency ultrasound. Administration of a high‑fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high‑fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high‑fat diet. A similar change was observed for the TJ proteins ZO‑1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ

  5. Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots.

    Directory of Open Access Journals (Sweden)

    Hector N Aguilar

    Full Text Available BACKGROUND: Quantification of phospho-proteins (PPs is crucial when studying cellular signaling pathways. Western immunoblotting (WB is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20 in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses. METHODOLOGY/PRINCIPAL FINDINGS: ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR scanner (Odyssey(R to quantify signals arising from near-infrared (NIR fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT-stimulated MLC(20 phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT. CONCLUSION/SIGNIFICANCE: ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW

  6. Myosin light chain kinase inhibitor ML7 improves vascular endothelial dysfunction via tight junction regulation in a rabbit model of atherosclerosis.

    Science.gov (United States)

    Cheng, Xiaowen; Wang, Xiaobian; Wan, Yufeng; Zhou, Qing; Zhu, Huaqing; Wang, Yuan

    2015-09-01

    Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet‑induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)‑1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high‑fat diet‑fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high‑fat diet (AS group) or a high‑fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium‑dependent relaxation and endothelium‑independent relaxation were measured using high-frequency ultrasound. Administration of a high‑fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high‑fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high‑fat diet. A similar change was observed for the TJ proteins ZO‑1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ

  7. Myosin light chain kinase is necessary for post-shock mesenteric lymph drainage enhancement of vascular reactivity and calcium sensitivity in hemorrhagic-shocked rats

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.P.; Niu, C.Y.; Zhao, Z.G.; Zhang, L.M.; Si, Y.H. [Institute of Microcirculation, Hebei North University, Hebei (China)

    2013-08-10

    Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca{sup 2+} were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca{sup 2+} at various concentrations. Maximum contractility (E{sub max}) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca{sup 2+} (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca{sup 2+} at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in E{sub max} for NE and from 0.729±0.037 to 0.645±0.056 g/mg in E{sub max} for Ca{sup 2+}, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.

  8. Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.

    Science.gov (United States)

    Fabian, Lacramioara; Troscianczuk, Joanna; Forer, Arthur

    2007-01-01

    Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. PMID:17381845

  9. The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.

    Science.gov (United States)

    Fusi, Luca; Huang, Zhe; Irving, Malcolm

    2015-08-18

    In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads. PMID:26287630

  10. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    OpenAIRE

    1987-01-01

    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the hea...

  11. The role of myosin phosphorylation in anaphase chromosome movement.

    Science.gov (United States)

    Sheykhani, Rozhan; Shirodkar, Purnata V; Forer, Arthur

    2013-01-01

    This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production. Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin. PMID:23566798

  12. 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

    2010-08-01

    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.

  13. Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division.

    Science.gov (United States)

    Silverman-Gavrila, Rosalind V; Forer, Arthur

    2003-06-01

    We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the

  14. 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.

  15. Recent Advances in Understanding Plant Myosin Function: Life in the Fast Lane

    Institute of Scientific and Technical Information of China (English)

    Imogen Sparkes

    2011-01-01

    Plant myosins are required for organelle movement,and a role in actin organization has recently come to light.Myosin mutants display several gross morphological phenotypes,the most severe being dwarfism and reduced fecundity,and there is a correlation between reduced organelle movement and morphological defects.This review aims to discuss recent findings in plants relating to the role of myosins in actin dynamics,development,and organelle movement,more specifically the endoplasmic reticulum (ER).One overarching theme is that there still appear to be more questions than answers relating to plant myosin function and regulation.

  16. Mechanism of Acetyl Salicylic Acid (Aspirin Degradation under Solar Light in Presence of a TiO2-Polymeric Film Photocatalyst

    Directory of Open Access Journals (Sweden)

    Debjani Mukherjee

    2016-04-01

    Full Text Available Application of titanium dioxide (TiO2 as a photocatalyst has presented a promising avenue for the safe photocatalytic degradation of pollutants. Increasing levels of the release of pharmaceuticals in the environment and formation of the intermediates during their degradation may impose health and environmental risks and therefore require more attention. Photocatalytic degradation of acetylsalicylic acid (aspirin was carried out in the presence of the TiO2-filled polymeric film as a photocatalyst under solar light irradiation. The polymeric film incorporates TiO2 in the matrix, which acts as a photocatalyst under solar illumination and degrades the acetyl salicylic acid (ASA into a range of organic compounds before complete demineralization (formation of carbon dioxide and water as final products. Among the intermediates, acetic acid was found to be present in a larger amount compared to other organic acids. The qualitative/quantitative analyses of the intermediates resulted in the determination of the most probable reaction’s mechanism in the degradation process. The mechanism of degradation of acetylsalicylic acid and its reaction pathway were developed from liquid chromatography/mass spectroscopy (LC/MS, Fourier Transform Infra Red (FTIR and UV spectrophotometric analysis. It was found that hydroxyl groups were dominant in the degradation process compared to electrons and holes generated by TiO2. The total organic carbon (TOC analysis was also carried out to analyze the organic carbon content of the intermediates formed during the course of degradation.

  17. Effect of Acetylation Treatment on Light Fastness and Thermal Stability of Pinus sylvestris var. mongolica Wood%乙酰化处理对樟子松木材耐光性和热稳定性的影响

    Institute of Scientific and Technical Information of China (English)

    郭洪武; 刘毅; 付展; 胡极航; 张帆

    2015-01-01

    【目的】探讨乙酰化处理对人工林木材耐光性和热稳定性的影响,为木材颜色调控技术及高耐光染色木材的研发提供理论依据。【方法】以樟子松木粉为试样,加入乙酸酐和二甲苯溶液,在120℃条件下分别反应5,10,20,40,60 min,测试乙酰化处理时间对木粉增重率的影响;分别称取1 g经不同时间乙酰化处理的木粉和未处理木粉,置于 UV老化试验箱内辐射100 h,利用红外光谱分析 UV辐射前后乙酰化木粉化学官能团的变化,通过热重和扫描电镜分析乙酰化木粉的热稳定性及其形貌变化。【结果】随着乙酰化处理时间的延长,樟子松木粉的增重率呈现先增加后降低的趋势,在处理40 min 时木粉增重率最大;乙酰化木粉在1741 cm -1和1385 cm -1处的CO,C—H特征吸收峰强度均大于原木粉,处理时间40 min 时木粉的吸收峰强度最大;UV 辐射后,乙酰化木粉在1508 cm -1处木质素苯环特征吸收峰强度明显大于原木粉,处理时间40 min 时木粉的吸收峰强度最大,表明木粉经乙酰化处理后光稳定性得到提升;热重分析显示,经乙酰化处理后,木粉热分解所需的温度明显提高,表明乙酰化木粉的热稳定性好于原木粉;扫描电镜分析表明,乙酰化处理可增强木粉微观构造抵抗光劣化的能力。【结论】乙酰化处理能有效抑制樟子松木材的光降解反应并提升其热稳定性。%Objective]Wood as well as wooden decorative materials produced by dyeing or/and color modulation are easy to be discoloration and degradation when exposed to light radiation. These will decrease its decorative effect and shorten the service life. The objective of this study was to investigate the effect of acetylation treatment on light fastness and thermal stability of plantation wood,and provide a theoretical basis for wood color regulation technology and the

  18. Involvement of myosin in intracellular motility and cytomorphogenesis in Micrasterias.

    Science.gov (United States)

    Oertel, Anke; Holzinger, Andreas; Lütz-Meindl, Ursula

    2003-01-01

    Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM. Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements. PMID:14642529

  19. Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes.

    Science.gov (United States)

    Fabian, Lacramioara; Forer, Arthur

    2007-01-01

    We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes. PMID:17922265

  20. 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

  1. Genetics Home Reference: myosin storage myopathy

    Science.gov (United States)

    ... myosin rod cause myosin storage myopathy via multiple mechanisms. Proc Natl Acad Sci U S A. 2009 Apr ... PubMed Tajsharghi H, Oldfors A. Myosinopathies: pathology and mechanisms. Acta Neuropathol. 2013 Jan;125(1):3-18. ...

  2. Myosin is involved in postmitotic cell spreading

    OpenAIRE

    1995-01-01

    We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading....

  3. 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.

  4. Species differences in the effects of prostaglandins on inositol trisphosphate accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cyclic AMP system.

    Science.gov (United States)

    Yousufzai, S Y; Chen, A L; Abdel-Latif, A A

    1988-12-01

    Comparative studies on the effects of prostaglandins (PGs) on 1,2-diacylglycerol, measured as phosphatidic acid (PA), and inositol trisphosphate (IP3) production, cyclic AMP (cAMP) formation, myosin light chain (MLC) phosphorylation and contraction in the iris sphincter smooth muscle of rabbit, bovine and other mammalian species were undertaken and functional and biochemical relationships between the IP3-Ca++ and cAMP second messenger systems were demonstrated. The findings obtained from these studies can be summarized as follows: 1) all PGs investigated, including PGE2, PGF2 alpha, PGF2 alpha-ester, PGE1 and PGA2 increased IP3 accumulation and PA formation, and the extent of stimulation was dependent on the animal species. Thus, PGF2 alpha-ester (1 microM), the most potent of the PGs, increased IP3 accumulation in rabbit and bovine sphincters by 33 and 58%, respectively, and increased PA formation by 67 and 56%, respectively. The PG increased IP3 accumulation in both rabbit and bovine sphincters very rapidly (T1/2 values about 26 sec) and in a dose-dependent manner. 2) The PG had no effect on MLC phosphorylation in the rabbit sphincter, but it increased that of the bovine by 36%. 3) The PG increased cAMP formation by 75% in the rabbit sphincter but it had no effect on that of the bovine. 4) The PG induced a maximal contractile response in the bovine sphincter but it had no effect on that of the rabbit. 5) In the bovine, PGA2 induced IP3 accumulation and contraction, without an effect on cAMP formation; however, in the rabbit, cat and dog it increased cAMP formation and had no effect on IP3 accumulation and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Sequential Myosin Phosphorylation Activates Tarantula Thick Filament via a Disorder-Order Transition

    OpenAIRE

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D.; Padrón, Raúl

    2015-01-01

    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscle and a secondary (modulatory) role in striated muscle, which is regulated by Ca2+ via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying f...

  6. Calcium and cargoes as regulators of myosin 5a activity

    International Nuclear Information System (INIS)

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins

  7. 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.

  8. 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.

  9. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins.

    Science.gov (United States)

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan

    2016-01-01

    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes. PMID:27597808

  10. 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.

  11. 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.

  12. 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

    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...... 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...

  13. The Role of Dietary Protein Intake and Resistance Training on Myosin Heavy Chain Expression

    Directory of Open Access Journals (Sweden)

    Willoughby Darryn S

    2004-12-01

    Full Text Available Abstract During resistance training the muscle undergoes many changes. Possibly the most profound and significant changes are those that occur in the muscles contractile proteins. Increases in these contractile proteins are one of the primary factors contributing to myofibrillar hypertrophy. The most abundant muscle protein is myosin, which comprises 25% of the total muscle protein. Due to the large amount of skeletal muscle that is composed of myosin, changes in this fiber may have profound effects on skeletal muscle size and strength. The myosin molecule is made up of 6 subunits, 2 very large heavy chains, and 4 smaller light chains. The myosin heavy chain (MHC accounts for 25–30% of all muscle proteins making its size an important factor in skeletal muscle growth. In conjunction with resistance training, dietary protein intake must be adequate to illicit positive adaptations. Although many studies have evaluated the role of dietary protein intake on skeletal muscle changes, few have evaluated the MHC specifically. Research has clearly defined the need for dietary protein and resistance training to facilitate positive changes in skeletal muscle. The purpose of this review was to evaluate the current literature on the effects of dietary protein and resistance training on the expression of the myosin heavy chain.

  14. 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.

  15. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek;

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  16. Preparation of human cardiac anti-myosin: a review

    International Nuclear Information System (INIS)

    The present communication is a review of the physicochemical characterization and immunological properties of myosin isolated from the cardiac muscle, the production of monoclonal antibody anti-myosin, the radiolabeling of this antibody and its applications as radiopharmaceuticals to imaging myocardial infarcts. The classical example of radioimmunologic diagnosis of non malignant tissues is the detection of myocardial infarction by radiolabeled antibodies to myosin. (author)

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize

    Directory of Open Access Journals (Sweden)

    Horst Ina

    2009-12-01

    Full Text Available Abstract Background Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant. Results We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm. Conclusion Our results

  2. Fitting of atomic coordinates of myosin S1 into the envelope of the 3-D reconstruction of muscle thick filaments

    International Nuclear Information System (INIS)

    Recently atomic coordinates of myosin S1of hen pectoral muscle have been reported (Rayment et al. Science 261: 50-58, 1993), allowing to know the precise position of the Regulatory Light Chain (RLC), the Essential Light Chain (ELC), as well as the interlacing places of ATP and actin. By means of the use of the Program of Advanced Three-dimensional Visualization AVS (Advanced Visual Systems, Inc., Waltham, M A, USA) we have been able to obtain the surface of the three-dimensional reconstruction of the thick filaments of tarantula muscle (Crowther et al. J. Mol. Biol. 184: 429-439, 1985) which shows a topographical detail associated to each myosin head (subfragment S1) non previously seen, and confirmed in a very evident way the antiparallel arrangement of both heads of a same myosin molecule. In view of the above-mentioned we have carried out an approximate adjustment of reported atomic coordinates of sub fragment S1 to the surface of one myosin head of the three-dimensional reconstruction. This adjustment allows to locate the approximate position of the Light Chains RLC and ELC, as well as the interlacing place of ATP and actin. The precise determination of the position of RLC and its phosphoryl able serine in the three-dimensional reconstruction can be important in terms of the molecular regulation mechanism of the muscular contraction bounded to the myosin that happens through the phosphorylation of RLC

  3. Purification and characterization of myosin from wheat mitochondria

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Myosin was purified from wheat mitochondria using DE-52 anion exchange chromatography and Sephacryl S-300 gel ffitration. The molecular weight of its heavy chain is about 210 ku, similar to that of muscle myosin Ⅱ (205 ku),and it could be recognized by the polyclonal antibodies against human skeletal muscle myosin Ⅱ. The ATPase activity of the mitochondrial myosin stimulated by F-actin from chicken muscle is 202.5 nmoles Pi/min @ mg. The mitochondrial myosin could be activated by Ca2+ and was not inhibited by Ca2+ at high concentration. The results demonstrate that the myosin of wheat mitochondria shares some similarities with the skeletal muscle myosin Ⅱ.

  4. Regulation of intermediary metabolism by protein acetylation

    OpenAIRE

    Guan, Kun-Liang; Xiong, Yue

    2010-01-01

    Extensive studies during the past four decades have identified important roles for lysine acetylation in the regulation of nuclear transcription. Recent proteomic analyses on protein acetylation uncovered a large number of acetylated proteins in the cytoplasm and mitochondria, including most enzymes involved in intermediate metabolism. Acetylation regulates metabolic enzymes by multiple mechanisms, including via enzymatic activation or inhibition, and by influencing protein stability. Convers...

  5. 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)

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Fatal Intoxication with Acetyl Fentanyl.

    Science.gov (United States)

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. PMID:26389815

  8. Nuclear myosin I regulates cell membrane tension

    Science.gov (United States)

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  9. The On-off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Himmel, D.; Mui, S; O& apos; Neall-Hennessey, E; Szent-Györgyi, A; Cohen, C

    2009-01-01

    In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long {alpha}-helical 'heavy chain' stabilized by a 'regulatory' light chain (RLC) and an 'essential' light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca{sup 2+} to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca{sup 2+}. Our results indicate that loss of Ca{sup 2+} abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.

  10. Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

    OpenAIRE

    Mun, Ji Young; Previs, Michael J.; Yu, Hope Y.; Gulick, James; Tobacman, Larry S.; Beck Previs, Samantha; Robbins, Jeffrey; Warshaw, David M.; Craig, Roger

    2014-01-01

    Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction. In the heart, MyBP-C modulates contractility in response to cardiac stimulation; mutations in MyBP-C lead to cardiac disease. The mechanism by which MyBP-C modulates cardiac contraction is not understood. Using electron microscopy and a light microscopic assay for filament sliding, we demonstrate that MyBP-C binds to the o...

  11. 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...

  12. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    Directory of Open Access Journals (Sweden)

    Cyr Janet L

    2002-11-01

    Full Text Available Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3 in its neck region; we identified a fourth IQ domain (IQ4, located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. Conclusions We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

  13. 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.

  14. Enhanced force generation by smooth muscle myosin in vitro.

    OpenAIRE

    VanBuren, P; Work, S S; Warshaw, D.M.

    1994-01-01

    To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was ...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin...... radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation...

  16. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

    International Nuclear Information System (INIS)

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

  17. Partial acetylation in situ of cellulosic fabric and its resistance to microbial and actinic degradation

    Directory of Open Access Journals (Sweden)

    K. L. Maheshwari

    1963-04-01

    Full Text Available Partial acetylation in situ of cotton fabrics can be achieved satisfactorily when it is carried out using 0.03 percent perchloricacid as the catalyst at a temperature not exceeding 30DegreeC. Increase in concentration of perchloric acid or temperature adversely affects the tensile strength of the fabrics. The acetylation of fabrics confers resistance to leaching. Scouring of the fabric prior to acetylation does not materially influence the extent of substitution of hydroxyl groups by acetyl ones. The acetylating mixture can be repeatedly used but fresh addition of the catalyst is necessary. With increase in the period of acetylation or in the quantity of catalyst, there is a progressive increase in acetic acid yield but the tensile strength of the fabric shows a corresponding decrease. Partial acetylation affords protection against microbial attack when the acetyl content of the acetylated fabrics is 14-15 percent. The treatment does not however afford protection against degradation by artificial light, or sunlight or weathering.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. 重组平滑肌肌球蛋白轻链激酶及ATP结合位点突变体的构建及表达%Construction and Expression of the Recombinant Smooth Muscle Myosin Light Chain Kinase and the ATP Binding Site Mutant

    Institute of Scientific and Technical Information of China (English)

    崔颖; 谢策; 魏晓晴; 赵莹; 吕广艳; 王辉; 高颖

    2009-01-01

    目的 构建重组全长平滑肌肌球蛋白轻链激酶(myosin light chain kinase,MLCK)ATP结合位点突变体,以研究平滑肌MLCK的结构与功能.方法 利用试剂盒对MLCK ATP结合位点进行定点突变,构建全长平滑肌MLCK ATP结合位点突变体重组表达载体pCold/BsMLCK/△ATP,在大肠杆菌中表达:利用SDS-PAGE鉴定表达的重组全长平滑肌MLCK ATP结合位点突变体在细胞中的分布:运用亲和层析及凝胶过滤分离纯化重组全长MLCK并利用SDS-PAGE鉴定表达MLCK的纯度.结果 重组全长MLCK/△ATP(突变型)在大肠杆菌中以可溶的形式大量表达;在样品的上清和沉淀中均有重组全长MLCK/△ATP表达;经CaM-Sepharose 4B和Superose 6 HR纯化,SDS-PAGE鉴定得到单一的表达条带.结论 重组全长MLCK/△ATP(突变型)可以在大肠杆菌中以可溶形式大量表达;SDS-PAGE结果显示重组全长MLCK/△ATP可以通过亲和层析和凝胶过滤纯化得到单一的条带.

  20. Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus.

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    Full Text Available KSHV is etiologically associated with Kaposi's sarcoma (KS, an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA, in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex

  1. Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Hoffmann, Else K; Pedersen, Stine F

    2007-01-01

    Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC......) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts......, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact...

  2. Protein Acetylation in Archaea, Bacteria, and Eukaryotes

    Directory of Open Access Journals (Sweden)

    Jörg Soppa

    2010-01-01

    Full Text Available Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which—Alba—was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  3. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    DEFF Research Database (Denmark)

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A;

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  4. Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

    Science.gov (United States)

    Forer, Arthur; Fabian, Lacramioara

    2005-04-01

    BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro. We discuss how the two sets of apparently contradictory results might be resolved, and we suggest possible experiments that might clarify the contradictory interpretations. PMID:15868207

  5. Myosins and cell dynamics in cellular slime molds.

    Science.gov (United States)

    Yumura, Shigehiko; Uyeda, Taro Q P

    2003-01-01

    Myosin is a mechanochemical transducer and serves as a motor for various motile activities such as cell migration, cytokinesis, maintenance of cell shape, phagocytosis, and morphogenesis. Nonmuscle myosin in vivo does not either stay static at specific subcellular regions or construct highly organized structures, such as sarcomere in skeletal muscle cells. The cellular slime mold Dictyostelium discoideum is an ideal "model organism" for the investigation of cell movement and cytokinesis. The advantages of this organism prompted researchers to carry out pioneering cell biological, biochemical, and molecular genetic studies on myosin II, which resulted in elucidation of many fundamental features of function and regulation of this most abundant molecular motor. Furthermore, recent molecular biological research has revealed that many unconventional myosins play various functions in vivo. In this article, how myosins are organized and regulated in a dynamic manner in Dictyostelium cells is reviewed and discussed. PMID:12722951

  6. Swelling of acetylated wood in organic liquids

    CERN Document Server

    Obataya, E; Obataya, Eiichi; Gril, Joseph

    2005-01-01

    To investigate the affinity of acetylated wood for organic liquids, Yezo spruce wood specimens were acetylated with acetic anhydride, and their swelling in various liquids were compared to those of untreated specimens. The acetylated wood was rapidly and remarkably swollen in aprotic organic liquids such as benzene and toluene in which the untreated wood was swollen only slightly and/or very slowly. On the other hand, the swelling of wood in water, ethylene glycol and alcohols remained unchanged or decreased by the acetylation. Consequently the maximum volume of wood swollen in organic liquids was always larger than that in water. The effect of acetylation on the maximum swollen volume of wood was greater in liquids having smaller solubility parameters. The easier penetration of aprotic organic liquids into the acetylated wood was considered to be due to the scission of hydrogen bonds among the amorphous wood constituents by the substitution of hydroxyl groups with hydrophobic acetyl groups.

  7. Flow properties of acetylated chickpea protein dispersions.

    Science.gov (United States)

    Liu, Li H; Hung, Tran V

    2010-06-01

    Chickpea protein concentrate was acetylated with acetic anhydride at 5 levels. Acetylated chickpea protein (ACP) dispersions at 3 levels (6%, 45%, and 49%) were chosen for this flow property study. Effects of protein concentration, temperature, concentrations of salt addition and particularly, degree of acetylation on these properties were examined. Compared with native chickpea proteins, the ACP dispersions exhibited a strong shear thinning behavior. Within measured temperature range (15 to 55 degrees C), the apparent viscosities of native chickpea protein dispersions were temperature independent; those of ACP dispersions were thermally affected. The flow index (n), consistency coefficient (m), apparent yield stress, and apparent viscosities of ACP dispersions increased progressively up to 45% acetylation but decreased at 49% acetylation level. Conformational studies by gel filtration suggested that chickpea proteins were associated or polymerized at up to 45% acetylation but the associated subunits gradually dissociated to smaller units at higher levels (49%) of acetylation.

  8. Histone Acetylation in Drug Addiction

    OpenAIRE

    Renthal, William; Nestler, Eric J.

    2009-01-01

    Regulation of chromatin structure through post-translational modifications of histones (e.g. acetylation) has emerged as an important mechanism to translate a variety of environmental stimuli, including drugs of abuse, into specific changes in gene expression. Since alterations in gene expression are thought to contribute to the development and maintenance of the addicted state, recent efforts are aimed at identifying how drugs of abuse alter chromatin structure and the enzymes which regulate...

  9. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    Science.gov (United States)

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

  10. Myosin-I Isozymes in Neonatal Rodent Auditory and Vestibular Epithelia

    OpenAIRE

    Dumont, Rachel A.; Zhao, Yi-Dong; Holt, Jeffrey R.; Bähler, Martin; Gillespie, Peter G.

    2002-01-01

    Myosin isozymes are essential for hair cells, the sensory cells of the inner ear. Because a myosin-I subfamily member may mediate adaptation of mechanoelectrical transduction, we examined expression of all eight myosin-I isozymes in rodent auditory and vestibular epithelia. Using RT-PCR, we found prominent expression of three isozymes, Myo1b (also known as myosin-Ia or myr 1), Myo1c (myosin-Ib or myr 2), and Myo1e (myr 3). By contrast, Myo1a (brush-border myosin-I), Myo1d (myosin lg or myr 4)...

  11. Different subcellular localizations and functions of Arabidopsis myosin VIII

    Directory of Open Access Journals (Sweden)

    Belausov Eduard

    2008-01-01

    Full Text Available Abstract Background Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. Results In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain, fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding. Conclusion Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.

  12. Full-length cDNA Cloning and Expression Analysis of Myosin Essential Light Chain Gene (MELC) from Hyriopsis cumingii%三角帆蚌肌球蛋白必需轻链基因(MELC)的cDNA全长克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    舒妙安; 胡杭娇; 陆晶莹; 徐宾朋; 王岩; 刘广绪; 郭晓令

    2013-01-01

    肌球蛋白必需轻链基因是EF-hand钙结合蛋白家族的成员,具有很强的Ca2+结合活性.为了研究三角帆蚌钙代谢基因调控机制,本研究采用同源克隆策略和RACE技术,从三角帆蚌外套膜组织中成功克隆得到肌球蛋白必需轻链基因(myosin essential light chain,MELC)的全长cDNA序列,共1119bp,开放阅读框468 bp(47~514),编码155个氨基酸,相对分子量为17.49 kD,理论等电点为4.53,基因序列提交GenBank的登录号为JX275828.通过PROSITE tools软件预测显示三角帆蚌MELC基因有一对保守的EF-hand结构域,可结合Ca2+,属于EF-hand钙结合蛋白家族.同源性比对分析显示,三角帆蚌MELC基因与合浦珠母贝(Pinctada fucata)、太平洋牡蛎(Crassostrea gigas)、盘鲍(Haliotis discus discus)等海水贝类的MELC基因同源性最高,依次为69%、69%和68%.经实时荧光定量PCR检测,发现MELC基因在闭壳肌、外套膜、鳃、斧足、性腺、心脏、肝脏、肠等8个组织中均有表达,其中在外套膜、鳃和斧足等与贝类钙代谢相关的组织中表达量远高于肝脏、肠等消化器官,预示MELC基因可能参与三角帆蚌的钙代谢过程.不同Ca2+浓度处理实验的结果表明,随着水体中Ca2+浓度逐渐升高,三角帆蚌MELC基因在外套膜中的表达量呈先上调后下降的趋势,在60 mg/L时达到最高值,表明适宜的Ca2+浓度可以刺激MELC基因大量表达,而过高或过低的Ca2+浓度均会抑制MELC基因表达.实验结果表明三角帆蚌MELC基因与Ca2+吸收、转运与贮存相关,可能参与珍珠等生物矿化过程.%Hyriopsis cumingii is an important pearl-breeding mussel in China,and it grows fast and breeds pearls of good quality.In this study,We cloned a full-length cDNA sequence of myosin essential light chain gene(MELC) which was isolated from the mantle of Hyriopsis cumingii according to homology cloning strategy and SMART RACE-PCR technique.The full length cDNA sequence

  13. A Method to determine lysine acetylation stoichiometries

    Energy Technology Data Exchange (ETDEWEB)

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  14. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    DEFF Research Database (Denmark)

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas;

    2015-01-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or...... suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of...... fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...

  15. Comparative analysis of pharmacological treatments with N-acetyl-dl-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    Science.gov (United States)

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. PMID:26607469

  16. 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.

  17. 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

  18. 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.

  19. Acetylation of Chinese bamboo flour and thermoplasticity

    Institute of Scientific and Technical Information of China (English)

    LI Xue-fang; CHEN Qin-hui; LIN Jin-huo; ZHUO Dong-xian; WU Xiu-ling

    2008-01-01

    Chinese bamboo flour was chemically modified by acetylation with acetic anhydride by using trichloroacetic acid as an activation agent and the optimized condition for acetylation of bamboo flour was determined as the trichloroacetic acid amount 6.0 g per 1.5-g bamboo flour, ultrasosonication duration 40 min and the reaction time 1 h at 65℃. The composition, microstructure and thermal behavior of acetylated bamboo flour were preliminarily characterized by FT-IR, DSC and SEM etc. The acetylated bamboo flour can be molded into sheets at 130℃ and 10 MPa, indicating the modified bamboo flour possesses thermalplastic performance.

  20. Acetylation of woody lignocellulose: significance and regulation

    Directory of Open Access Journals (Sweden)

    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  1. Acetylation regulates Jun protein turnover in Drosophila.

    Science.gov (United States)

    Zhang, Daoyong; Suganuma, Tamaki; Workman, Jerry L

    2013-11-01

    C-Jun is a major transcription factor belonging to the activating protein 1 (AP-1) family. Phosphorylation has been shown to be critical for c-Jun activation and stability. Here, we report that Jra, the Drosophila Jun protein, is acetylated in vivo. We demonstrate that the acetylation of Jra leads to its rapid degradation in response to osmotic stress. Intriguingly, we also found that Jra phosphorylation antagonized its acetylation, indicating the opposite roles of acetylation and phosphorylation in Jra degradation process under osmotic stress. Our results provide new insights into how c-Jun proteins are precisely regulated by the interplay of different posttranslational modifications.

  2. Acetylation-Mediated Suppression of Transcription-Independent Memory: Bidirectional Modulation of Memory by Acetylation

    OpenAIRE

    Katja Merschbaecher; Jakob Haettig; Uli Mueller

    2012-01-01

    Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs), and the antagonistic histone deacetylases (HDACs) play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM). While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact...

  3. Internal Motility in Stiffening Actin-Myosin Networks

    OpenAIRE

    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 sug...

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

    OpenAIRE

    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

    2008-01-01

    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 Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence...

  5. A Perspective on the Role of Myosins as Mechanosensors.

    Science.gov (United States)

    Greenberg, Michael J; Arpağ, Göker; Tüzel, Erkan; Ostap, E Michael

    2016-06-21

    Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms. PMID:27332116

  6. 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

  7. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    Science.gov (United States)

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  8. Investigation of acetyl migrations in furanosides

    Directory of Open Access Journals (Sweden)

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  9. The Acetylation of Starch by Reactive Extrusion

    NARCIS (Netherlands)

    Graaf, Robbert A. de; Broekroelofs, Annet; Janssen, Léon P.B.M.

    1998-01-01

    Potato starch has been acetylated in a counter rotating twin screw extruder using vinylacetate and sodium hydroxide. The desired starch acetylation reaction is accompanied by an undesired parallel base catalysed hydrolysis reaction of vinylacetate and a consecutive hydrolysis reaction of the acetyla

  10. Acetylation Enhances the Promoting Role of AIB1 in Breast Cancer Cell Proliferation

    Science.gov (United States)

    You, Dingyun; Zhao, Hongbo; Wang, Yan; Jiao, Yang; Lu, Minnan; Yan, Shan

    2016-01-01

    The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator, which is overexpressed in various types of human cancers, including breast cancer. However, the molecular mechanisms regulating AIB1 function remain largely unknown. In this study, we present evidence demonstrating that AIB1 is acetylated by MOF in human breast cancer cells. Moreover, we also found that the acetylation of AIB1 enhances its function in promoting breast cancer cell proliferation. We further showed that the acetylation of AIB1 is required for its recruitment to E2F1 target genes by E2F1. More importantly, we found that the acetylation levels of AIB1 are greatly elevated in human breast cancer cells compared with that in non-cancerous cells. Collectively, our results shed light on the molecular mechanisms that regulate AIB1 function in breast cancer. PMID:27665502

  11. 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.

  12. Histone Acetylation in Fungal Pathogens of Plants

    Directory of Open Access Journals (Sweden)

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  13. Light

    CERN Document Server

    Robertson, William C

    2003-01-01

    Why is left right and right left in the mirror? Baffled by the basics of reflection and refraction? Wondering just how the eye works? If you have trouble teaching concepts about light that you don t fully grasp yourself, get help from a book that s both scientifically accurate and entertaining with Light. By combining clear explanations, clever drawings, and activities that use easy-to-find materials, this book covers what science teachers and parents need to know to teach about light with confidence. It uses ray, wave, and particle models of light to explain the basics of reflection and refraction, optical instruments, polarization of light, and interference and diffraction. There s also an entire chapter on how the eye works. Each chapter ends with a Summary and Applications section that reinforces concepts with everyday examples. Whether you need a deeper understanding of how light bends or a good explanation of why the sky is blue, you ll find Light more illuminating and accessible than a college textbook...

  14. Elastic lever arm model for myosin V

    CERN Document Server

    Vilfan, A

    2005-01-01

    We present a mechanochemical model for myosin V, a two-headed processive motor protein. We derive the properties of a dimer from those of an individual head, which we model both with a 4-state cycle (detached, attached with ADP.Pi, attached with ADP and attached without nucleotide) and alternatively with a 5-state cycle (where the power stroke is not tightly coupled to the phosphate release). In each state the lever arm leaves the head at a different, but fixed, angle. The lever arm itself is described as an elastic rod. The chemical cycles of both heads are coordinated exclusively by the mechanical connection between the two lever arms. The model explains head coordination by showing that the lead head only binds to actin after the power stroke in the trail head and that it only undergoes its power stroke after the trail head unbinds from actin. Both models (4- and 5-state) reproduce the observed hand-over-hand motion and fit the measured force-velocity relations. The main difference between the two models c...

  15. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Directory of Open Access Journals (Sweden)

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

  16. Two Regions of the Tail Are Necessary for the Isoform-specific Functions of Nonmuscle Myosin IIB

    OpenAIRE

    Sato, Masaaki K.; Takahashi, Masayuki; Yazawa, Michio

    2007-01-01

    To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing a...

  17. 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.

  18. 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.

  19. Simultaneous quantification of differently glycosylated, acetylated and 2,3-dihydro-2,5-dihydroxy-6-methyl-4h-pyran-4-one-conjugated soyasaponins using reversed-phase high-performance liquid chromatography with evaporative light scattering detection

    NARCIS (Netherlands)

    DeCroos, K.; Vincken, J.P.; Heng, L.; Bakker, R.; Gruppen, H.; Verstraete, W.

    2005-01-01

    A novel method utilizing high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and electrospray ionisation mass spectrometry (ESI-MS) was developed for the analysis of soyasaponins, a divers group of triterpenic compounds with one or two sugar side chains,

  20. Light

    CERN Document Server

    Ditchburn, R W

    2011-01-01

    This classic study, available for the first time in paperback, clearly demonstrates how quantum theory is a natural development of wave theory, and how these two theories, once thought to be irreconcilable, together comprise a single valid theory of light. Aimed at students with an intermediate-level knowledge of physics, the book first offers a historical introduction to the subject, then covers topics such as wave theory, interference, diffraction, Huygens' Principle, Fermat's Principle, and the accuracy of optical measurements. Additional topics include the velocity of light, relativistic o

  1. Sequential myosin phosphorylation activates tarantula thick filament via a disorder-order transition.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D; Padrón, Raúl

    2015-08-01

    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscles and a secondary (modulatory) role in striated muscles, which is regulated by Ca(2+)via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca(2+)4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectory analysis revealed that an inter-domain salt bridge network (R39/E58,E61) facilitates the formation of a stable helix-coil-helix (HCH) motif formed by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of the blocked head on Ser45 does not induce any substantial structural changes. However, phosphorylation of the free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH folding, phosphorylation of Ser35 unlocks a compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by the subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition. PMID

  2. Review: The ATPase mechanism of myosin and actomyosin.

    Science.gov (United States)

    Geeves, Michael A

    2016-08-01

    Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016. PMID:27061920

  3. Efficient acetylation of primary amines and amino acids in environmentally benign brine solution using acetyl chloride

    Indian Academy of Sciences (India)

    Kaushik Basu; Suchandra Chakraborty; Achintya Kumar Sarkar; Chandan Saha

    2013-05-01

    Acetyl chloride is one of the most commonly available and cheap acylating agent but its high reactivity and concomitant instability in water precludes its use to carry out acetylation in aqueous medium. The present methodology illustrates the efficient acetylation of primary amines and amino acids in brine solution by means of acetyl chloride under weakly basic condition in the presence of sodium acetate and/or triethyl amine followed by trituration with aqueous saturated bicarbonate solution. This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the amide derivatives. Mechanistic rationale of this methodology is also important.

  4. 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.

  5. Nonmuscle myosin dependent synthesis of type I collagen

    OpenAIRE

    Cai, Le; Fritz, Dillon; Stefanovic, Lela; Stefanovic, Branko

    2010-01-01

    Type I collagen is the most abundant protein in human body synthesized in all tissues as the heterotrimer of two α1(I) and one α2(I) polypeptides. Here we show that intact nonmuscle myosin filaments are required for synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds RNA binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle my...

  6. 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.

  7. Levels of histone acetylation in thyroid tumors.

    Science.gov (United States)

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  8. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    Science.gov (United States)

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  9. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  10. 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.

  11. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  12. 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...

  13. 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.

  14. Model of Rho-Mediated Myosin Recruitment to the Cleavage Furrow during Cytokinesis

    Science.gov (United States)

    Veksler, Alexander; Vavylonis, Dimitrios

    2010-03-01

    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During cytokinesis, the myosin attached to the cell's cortex progressively disassembles at the flanking regions and concentrates in the equator [1]. This recruitment depends on myosin motor activity and activation by Rho proteins. Central spindle and astral microtubules establish a spatial pattern of differential Rho activity [2]. We propose a reaction-diffusion model for the dynamics of myosin and Rho proteins during cytokinesis. In the model, the mitotic spindle activates Rho at the equator. Active Rho promotes, in a switch-like manner, myosin assembly into cortical minifilaments. Mechanical stress by cortical myosin causes disassembly of myosin minifilaments and deactivates Rho. Our results explain both the recruitment of myosin to the cleavage furrow and the observed damped myosin oscillations in the cell's flanking regions [1]. Spatial extent, period and decay rate of myosin oscillations are calculated. Various regimes of myosin recruitment are predicted. [1] Zhou & Wang, Mol. Biol. Cell 19:318 (2008) [2] Murthy & Wadsworth, J. Cell Sci. 121:2350 (2008)

  15. Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

    Directory of Open Access Journals (Sweden)

    Khalid Amari

    2014-10-01

    Full Text Available Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP and to a delay in the MP accumulation in plasmodesmata (PD. The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

  16. 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.

  17. 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. PMID:26842936

  18. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    DEFF Research Database (Denmark)

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.;

    2014-01-01

    Results The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most promin...

  19. Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.

    Science.gov (United States)

    Alamo, Lorenzo; Qi, Dan; Wriggers, Willy; Pinto, Antonio; Zhu, Jingui; Bilbao, Aivett; Gillilan, Richard E; Hu, Songnian; Padrón, Raúl

    2016-03-27

    Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles. PMID:26851071

  20. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

    DEFF Research Database (Denmark)

    Schwer, Bjoern; Bunkenborg, Jakob; Verdin, Regis O;

    2006-01-01

    by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity......We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2...

  1. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    Science.gov (United States)

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.

  2. Quantitative determination of type I myosin heavy chain in bovine muscle with anti myosin monoclonal antibodies.

    Science.gov (United States)

    Picard, B; Leger, J; Robelin, J

    1994-01-01

    Bovine type I muscle fibers were characterized by enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for slow myosin heavy chains (MHC 1). Two bovine muscles, the Masseter and Cutaneus trunci, were analyzed by different complementary techniques: electrophoresis, immunoblotting and immunohistiology. The results showed that the two muscles have extreme characteristics. The Masseter contains only slow MHC and the Cutaneus trunci is composed solely of rapid MHC (MHC 2a and 2b). A standard for this ELISA was obtained by mixing the two muscles and was used as a reference in the determination of the percentage of MHC 1 in a given muscle. In this study, the Longissimus thoracis of 27 Charolais cattle were examined. The different conditions under which assays were carried out were described and the accuracy of the measurement was calculated. In view of the results, ELISA was chosen for the analysis of muscle fiber types in large numbers of animal specimens. This technique could be used in several research projects to study the muscle characteristics that determine beef quality. PMID:22061628

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

    Science.gov (United States)

    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.

  4. 21 CFR 172.828 - Acetylated monoglycerides.

    Science.gov (United States)

    2010-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  5. Acetylated flavonol triglycosides from Ammi majus L.

    Science.gov (United States)

    Singab, A N

    1998-12-01

    Two new acetylated flavonol triglycosides: kaempferol and isorhamnetin 3-O-[2"-(4"'-acetylrhamnosyl)-6"-glucosyl] glucosides, were isolated and identified from the aerial parts of Ammi majus L. In addition, three known flavonol glycosides namely; isorhamnetin-3-O-rutinoside, kaempferol-3-O-glucoside and isorhamnetin-3-O-glucoside were detected.

  6. Oxidative Debenzylation and Acetylation of Hexabenzylhexaazaisowutzitane

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The oxidative reactivity of hexabenzylhexaazaisowutzitane(HBIW)under different conditions was studied. It was found that the N-benzyl groups were found to form benzoyl group after oxidation. They might also be first debenzylated and then acetylated by potassium permanganate in acetic anhydride/DMF.

  7. Mode coupling points to functionally important residues in myosin II.

    OpenAIRE

    Varol, Onur; Yüret, Deniz; Erman, Burak; Kabakçıoğlu, Alkan

    2015-01-01

    Relevance of mode coupling to energy/information transfer during protein function, particularly in the context of allosteric interactions is widely accepted. However, existing evidence in favor of this hypothesis comes essentially from model systems. We here report a novel formal analysis of the near-native dynamics of myosin II, which allows us to explore the impact of the interaction between possibly non-Gaussian vibrational modes on fluctutational dynamics. We show that an information-theo...

  8. 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.

  9. Bighead carp myosin stability tb heat and frozen storage

    OpenAIRE

    Radičević Tatjana; Raičević Smiljana; Niketić Vesna P.

    2002-01-01

    Differential scanning calorimetry (DSC) was used to investigate thermal transitions of bighead carp muscle (Aristichthys nobilis Richardson). Three endothermic peaks were observed in DSC thermograms of fresh muscle. After addition of salt, transition temperatures shifted to lower temperatures. Preheating samples at 70°C caused virtual disappearance of all transition peaks. Low temperature storage (-18°C, for five weeks) caused changes in myosin transitions. The evidence suggests that changes ...

  10. Smooth muscle myosin: a high force-generating molecular motor.

    OpenAIRE

    VanBuren, P; Guilford, W. H.; Kennedy, G.; Wu, J.; Warshaw, D.M.

    1995-01-01

    Smooth muscle generates as much force per cross sectional area of muscle as skeletal muscle with only one-fifth the myosin content. Although this apparent difference could be explained at the tissue or cellular level, it is possible that at the molecular level smooth muscle cross-bridges generate greater average force than skeletal muscle cross-bridges. To test this hypothesis, we used an in vitro motility assay (VanBuren et al., 1994) in which either chicken thiophosphorylated gizzard smooth...

  11. 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.

  12. Immunoblot detection of Myosin and Myosin-Light-Chain-Kinase in trabecular meshwork cells of the eye

    OpenAIRE

    Schlott, Sebastian

    2010-01-01

    Glaucoma is characterised by inbalance of intraocular pressure (IOP) and optic nerve resistance. Findings comprise excavation of the optic nerve disk and characteristic visual field defects (glaucomatous neuropathy of the optic nerve). In industrialised countries glaucoma is the third commonest cause of blindness after age related macula degeneration and diabetes mellitus. Excessive IOP is one of the most important risk factors for optic nerve damage and represents the only avenue for therape...

  13. 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

  14. Structural basis for myopathic defects engendered by alterations in the myosin rod

    OpenAIRE

    Cammarato, Anthony; Li, Xiaochuan; Reedy, Mary C.; Lee, Chi F.; Lehman, William; Bernstein, Sanford I

    2011-01-01

    While mutations in the myosin S1 motor domain can directly disrupt the generation and transmission of force along myofibrils and lead to myopathy, the mechanism whereby mutations in the myosin rod influence mechanical function is less clear. Here, we used a combination of various imaging techniques and molecular dynamics simulations to test the hypothesis that perturbations in the myosin rod can disturb normal sarcomeric uniformity and, like motor domain lesions, would influence force product...

  15. Two New Acetyl Cimicifugosides from the Rhizomes of Cimicifuga Racemosa

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Two new acetyl cimicifugosides were isolated from the rhizomes of Cimicifuga racemosa. Their structures were elucidated as 2'-O-acetyl cimicifugoside H-1 1 and 3'-O-acetyl cimicifugoside H-1 2 by the spectroscopic evidence and chemical methods.

  16. Arabidopsis myosin XI-K localizes to the motile endomembrane vesicles associated with F-actin

    Directory of Open Access Journals (Sweden)

    Valera V. Peremyslov

    2012-09-01

    Full Text Available Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile ER subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Unconventional myosins, actin dynamics and endocytosis: a ménage à trois?

    Science.gov (United States)

    Soldati, Thierry

    2003-06-01

    Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation. PMID:12753645

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Myosin individualized: single nucleotide polymorphisms in energy transduction

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Interactions between N-acetyl-L-cysteine protected CdTe quantum dots and doxorubicin through spectroscopic method

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiupei, E-mail: xiupeiyang@163.com [Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, Nanchong 637000 (China); College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000 (China); Lin, Jia; Liao, Xiulin; Zong, Yingying; Gao, Huanhuan [College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000 (China)

    2015-06-15

    Highlights: • CdTe quantum dots with the diameter of 3–5 nm were synthesized in aqueous solution. • The modified CdTe quantum dots showed well fluorescence properties. • The interaction between the CdTe quantum dots and doxorubicin (DR) was investigated. - Abstract: N-acetyl-L-cysteine protected cadmium telluride quantum dots with a diameter of 3–5 nm were synthesized in aqueous solution. The interaction between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin was investigated by ultraviolet–visible absorption and fluorescence spectroscopy at physiological conditions (pH 7.2, 37 °C). The results indicate that electron transfer has occurred between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin under light illumination. The quantum dots react readily with doxorubicin to form a N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex via electrostatic attraction between the −NH{sub 3}{sup +} moiety of doxorubicin and the −COO{sup −} moiety of N-acetyl-L-cysteine/cadmium telluride quantum dots. The interaction of N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex with bovine serum albumin was studied as well, showing that the complex might induce the conformation change of bovine serum due to changes in microenvironment of bovine serum.

  3. Acetylation Is Indispensable for p53 Activation

    OpenAIRE

    Tang, Yi; Zhao, Wenhui; Chen, Yue; Zhao, Yingming; Gu, Wei

    2008-01-01

    The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its...

  4. p53 Acetylation: Regulation and Consequences

    OpenAIRE

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo ev...

  5. Fragrance material review on acetyl cedrene.

    Science.gov (United States)

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23907023

  6. Laing early onset distal myopathy: slow myosin defect with variable abnormalities on muscle biopsy

    NARCIS (Netherlands)

    P.J. Lamont; B. Udd; F.L. Mastaglia; M. de Visser; P. Hedera; T. Voit; L.R. Bridges; V. Fabian; A. Rozemuller; N.G. Laing

    2006-01-01

    Background: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7 It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one for

  7. 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.

  8. 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.

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

    OpenAIRE

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

    2016-01-01

    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.

  10. Intra-axonal myosin and actin in nerve regeneration.

    Science.gov (United States)

    McQuarrie, Irvine G; Lund, Linda M

    2009-10-01

    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  11. The energetics of allosteric regulation of ADP release from myosin heads.

    Science.gov (United States)

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  12. Acetylation phenotype variation in pediatric patients with atopic dermatitis

    Directory of Open Access Journals (Sweden)

    Rafi A Majeed Al-Razzuqi

    2011-01-01

    Full Text Available Background: Few studies have been done on the relation between acetylator status and allergic diseases. Aim: To determine any possible association between acetylating phenotype in pediatric patients with atopic dermatitis (AD and the disease prognosis. Patients and Methods: Thirty-six pediatric patients and forty two healthy children as a control group were participated in the study. All participants received a single oral dose of dapsone of 1.54 mg/kg body weight, after an overnight fast. Using high performance liquid chromatography (HPLC, plasma concentrations of dapsone and its metabolite (monoacetyldapsone were estimated to phenotype the participants as slow and rapid acetylators according to their acetylation ratio (ratio of monoacetyldapsone to dapsone. Results: 72.2% of pediatric patients with AD showed slow acetylating status as compared to 69.4% of control individuals. Also, 73% of AD patients with slow acetylating phenotype had familial history of allergy. The severity of AD occurred only in slow acetylator patients. The eczematous lesions in slow acetylators presented mainly in the limbs, while in rapid acetylators, they were found mostly in face and neck. Conclusion: This study shows an association between the N-acetylation phenotype variation and clinical aspects of AD.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Minimum energy reaction profiles for ATP hydrolysis in myosin.

    Science.gov (United States)

    Grigorenko, Bella L; Kaliman, Ilya A; Nemukhin, Alexander V

    2011-11-01

    The minimum energy reaction profiles corresponding to two possible reaction mechanisms of adenosine triphosphate (ATP) hydrolysis in myosin are computed in this work within the framework of the quantum mechanics-molecular mechanics (QM/MM) method by using the same partitioning of the model system to the QM and MM parts and the same computational protocol. On the first reaction route, one water molecule performs nucleophilic attack at the phosphorus center P(γ) from ATP while the second water molecule in the closed protein cleft serves as a catalytic base assisted by the Glu residue from the myosin salt bridge. According to the present QM/MM calculations consistent with the results of kinetic studies this reaction pathway is characterized by a low activation energy barrier about 10 kcal/mol. The computed activation energy barrier for the second mechanism, which assumes the penta-coordinated oxyphosphorane transition state upon involvement of single water molecule in the reaction, is considerably higher than that for the two-water mechanism. PMID:21839658

  16. Autoantibody germ-line gene segment encodes V{sub H} and V{sub L} regions of a human anti-streptococcal monoclonal antibody recognizing streptococcal M protein and human cardiac myosin epitopes

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, A.; Cunningham, M.W. [Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Adderson, E.E. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    1995-04-15

    Cross-reactivity of anti-streptococcal Abs with human cardiac myosin may result in sequelae following group A streptococcal infections. Molecular mimicry between group A streptococcal M protein and cardiac myosin may be the basis for the immunologic cross-reactivity. In this study, a cross-reactive human anti-streptococcal/antimyosin mAb (10.2.3) was characterized, and the myosin epitopes were recognized by the Ab identified. mAb 10.2.3 reacted with four peptides from the light meromyosin (LMM) tail fragment of human cardiac myosin, including LMM-10 (1411-1428), LMM-23 (1580-1597), LMM-27 (1632-1649), and LMM-30 (1671-1687). Only LMM-30 inhibited binding of mAb 10.2.3 to streptococcal M protein and human cardiac myosin. Human mAb 10.2.3 labeled cytoskeletal structures within rat heart cells in indirect immunofluorescence, and reacted with group A streptococci expressing various M protein serotypes, PepM5, and recombinant M protein. The nucleotide sequence of gene segments encoding the Ig heavy and light chain V region of mAb 10.2.3 was determined. The light chain V segment was encoded by a VK1 gene segment that was 98.5% identical with germ-line gene humig{sub K}Vi5. The V segment of the heavy chain was encoded by a V{sub H}3a gene segment that differed from the V{sub H}26 germ-line gene by a single base change. V{sub H}26 is expressed preferentially in early development and encodes autoantibodies with anti-DNA and rheumatoid factor specificities. Anti-streptococcal mAb 10.2.3 is an autoantibody encoded by V{sub H} and V{sub L} genes, with little or no somatic mutation. 63 refs., 11 figs.

  17. Determination of amphetamine by HPLC after acetylation.

    Science.gov (United States)

    Veress, T

    2000-01-01

    An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market. PMID:10641931

  18. Getting a Knack for NAC: N-Acetyl-Cysteine

    OpenAIRE

    Sansone, Randy A.; Sansone, Lori A.

    2011-01-01

    N-acetyl-cysteine, N-acetylcysteine, N-acetyl cysteine, and N-acetyl-L-cysteine are all designations for the same compound, which is abbreviated as NAC. NAC is a precursor to the amino acid cysteine, which ultimately plays two key metabolic roles. Through its metabolic contribution to glutathione production, cysteine participates in the general antioxidant activities of the body. Through its role as a modulator of the glutamatergic system, cysteine influences the reward-reinforcement pathway....

  19. Obesity, cancer, and acetyl-CoA metabolism

    OpenAIRE

    Lee, Joyce V.; Shah, Supriya A.; Wellen, Kathryn E.

    2013-01-01

    As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in r...

  20. Acetylation of Tau Inhibits Its Degradation and Contributes to Tauopathy

    OpenAIRE

    Min, Sang-Won; Cho, Seo-Hyun; Zhou, Yungui; Schroeder, Sebastian; Haroutunian, Vahram; Seeley, William W.; Huang, Eric J.; Shen, Yong; Masliah, Eliezer; Mukherjee, Chandrani; Meyers, David; Cole, Philip A.; Ott, Melanie; Gan, Li

    2010-01-01

    Neurodegenerative tauopathies characterized by hyperphosphorylated tau include frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). Reducing tau levels improves cognitive function in mouse models of AD and FTDP-17, but the mechanisms regulating the turnover of pathogenic tau are unknown. We found that tau is acetylated and that tau acetylation prevents degradation of phosphorylated tau (p-tau). Using two antibodies specific for acetylated ta...

  1. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Science.gov (United States)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  2. Protein lysine acetylation in bacteria: Current state of the art.

    Science.gov (United States)

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  3. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway.

    Science.gov (United States)

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang; McCrae, Keith R

    2013-11-28

    The antiphospholipid syndrome is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLAs). Most pathogenic APLAs are directed against β2-glycoprotein I (β2GPI), a plasma phospholipid binding protein. One mechanism by which circulating antiphospholipid/anti-β2GPI antibodies may promote thrombosis is by inducing the release of procoagulant microparticles from endothelial cells. However, there is no information available concerning the mechanisms by which anti-β2GPI antibodies induce microparticle release. In seeking to identify proteins phosphorylated during anti-β2GPI antibody-induced endothelial activation, we observed phosphorylation of nonmuscle myosin II regulatory light chain (RLC), which regulates cytoskeletal assembly. In parallel, we observed a dramatic increase in the formation of filamentous actin, a two- to fivefold increase in the release of endothelial cell microparticles, and a 10- to 15-fold increase in the expression of E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and tissue factor messenger RNA. Microparticle release, but not endothelial cell surface E-selectin expression, was blocked by inhibiting RLC phosphorylation or nonmuscle myosin II motor activity. These results suggest that distinct pathways, some of which mediate cytoskeletal assembly, regulate the endothelial cell response to anti-β2GPI antibodies. Inhibition of nonmuscle myosin II activation may provide a novel approach for inhibiting microparticle release by endothelial cells in response to anti-β2GPI antibodies. PMID:23954892

  4. 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.

  5. Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium.

    Science.gov (United States)

    Uyeda, T Q; Yumura, S

    2000-04-15

    The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. PMID:10816252

  6. Epithelial cell shape is regulated by Lulu proteins via myosin-II.

    Science.gov (United States)

    Nakajima, Hiroyuki; Tanoue, Takuji

    2010-02-15

    Cell-shape change in epithelial structures is fundamental to animal morphogenesis. Recent studies identified myosin-II as the major generator of driving forces for cell-shape changes during morphogenesis. Lulu (Epb41l5) is a major regulator of morphogenesis, although the downstream molecular and cellular mechanisms remain obscure in mammals. In Drosophila and zebrafish, Lulu proteins were reported to negatively regulate Crumbs, an apical domain regulator, thus regulating morphogenesis. In this study, we show that mammalian Lulu activates myosin-II, thus regulating epithelial cell shape. In our experiments, Lulu expression in epithelial cells resulted in apical constriction and lateral elongation in the cells, accompanied by upregulation of myosin-II. The inhibition of myosin-II activity almost completely blocked this Lulu-driven cell-shape change. We further found that Rock participates in the myosin-II activation. Additionally, RNAi-mediated depletion of Lulu in epithelial cells resulted in disorganization of myosin-II and a concomitant loss of proper lateral domain organization in the cells. From these results, we propose that Lulu regulates epithelial cell shape by controlling myosin-II activity. PMID:20103536

  7. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    Science.gov (United States)

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia

    2012-08-10

    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  8. The Accommodative Function of Myosin on Cell Biomechanics Effect%肌球蛋白在生物力学效应中的调控作用

    Institute of Scientific and Technical Information of China (English)

    胡鸣(综述); 洪莉(审校)

    2015-01-01

    Cytoskeleton is a component of mechanical force transmission chain .As the main protein in forming cytoskeleton in cells, myosin plays a role in regulating the effect produced by cells when they are forced by the external force.When the expression, structure and activity of myosin changes, mechanical effi-ciency of cells will have a corresponding change, thus affecting the cell function and organizational structure. Phosphorylated myosin light chain, transformation of subtypes in myosin heavy chain , and Rho GTPases sig-naling pathway play an important role in the regulation of cellular biological effects .In this article, we try to express the advanced research in myosin′s impact on cell biomechanics effect.In addition, we also discuss the generation mechanism of such effects.%细胞骨架是细胞内机械力传递链的一个组分,肌球蛋白作为细胞骨架的主要组成蛋白,对细胞受到外界力作用时产生的效应具有一定的调控作用,当细胞内的肌球蛋白的表达、结构以及活性发生改变时,细胞的力学效能也会发生相应的改变,从而影响细胞的功能以及组织结构的改变。肌球蛋白轻链的磷酸化、重链各亚型间的转化以及Rho GTP酶信号通路在对细胞生物力学效应的调控中起着一定的作用。

  9. 肌球蛋白在生物力学效应中的调控作用%The Accommodative Function of Myosin on Cell Biomechanics Effect

    Institute of Scientific and Technical Information of China (English)

    胡鸣(综述); 洪莉(审校)

    2015-01-01

    细胞骨架是细胞内机械力传递链的一个组分,肌球蛋白作为细胞骨架的主要组成蛋白,对细胞受到外界力作用时产生的效应具有一定的调控作用,当细胞内的肌球蛋白的表达、结构以及活性发生改变时,细胞的力学效能也会发生相应的改变,从而影响细胞的功能以及组织结构的改变。肌球蛋白轻链的磷酸化、重链各亚型间的转化以及Rho GTP酶信号通路在对细胞生物力学效应的调控中起着一定的作用。%Cytoskeleton is a component of mechanical force transmission chain .As the main protein in forming cytoskeleton in cells, myosin plays a role in regulating the effect produced by cells when they are forced by the external force.When the expression, structure and activity of myosin changes, mechanical effi-ciency of cells will have a corresponding change, thus affecting the cell function and organizational structure. Phosphorylated myosin light chain, transformation of subtypes in myosin heavy chain , and Rho GTPases sig-naling pathway play an important role in the regulation of cellular biological effects .In this article, we try to express the advanced research in myosin′s impact on cell biomechanics effect.In addition, we also discuss the generation mechanism of such effects.

  10. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes.

    Science.gov (United States)

    Prokesch, A; Pelzmann, H J; Pessentheiner, A R; Huber, K; Madreiter-Sokolowski, C T; Drougard, A; Schittmayer, M; Kolb, D; Magnes, C; Trausinger, G; Graier, W F; Birner-Gruenberger, R; Pospisilik, J A; Bogner-Strauss, J G

    2016-04-05

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes.

  11. The SAH domain extends the functional length of the myosin lever

    OpenAIRE

    Baboolal, TG; Sakamoto, T.; Forgacs, E; White, HD; Jackson, SM; Takagi, Y.; Farrow, RE; Molloy, JE; Knight, PJ; Sellers, JS; Peckham, M.

    2009-01-01

    Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to...

  12. 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.

  13. 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. PMID:27498562

  14. 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.

  15. The kinetics of the acetylation of gelatinised potato starch

    NARCIS (Netherlands)

    de Graaf, R.A.; Broekroelofs, G.A.; Janssen, L.P.B.M.; Beenackers, A.A C M

    1995-01-01

    The reaction rates, in the base-catalysed acetylation of gelatinised aqueous starch (4 wt%), by vinylacetate (ViAc), were investigated in a semibatch reactor at temperatures ranging from 20 to 50 degrees C. The desired starch acetylation reaction is accompanied by an undesired parallel base-catalyse

  16. Regulation of autophagy by cytosolic acetyl-coenzyme A

    DEFF Research Database (Denmark)

    Mariño, Guillermo; Pietrocola, Federico; Eisenberg, Tobias;

    2014-01-01

    Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic p...

  17. Medial temporal N-acetyl-aspartate in pediatric major depression.

    Science.gov (United States)

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  18. Medial temporal N-acetyl aspartate in pediatric major depression

    Science.gov (United States)

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  19. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Science.gov (United States)

    2010-04-01

    ... HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L... section. The minimum amount of the additive to achieve the desired effect must be used, and the...

  20. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a rev

  1. A facile and practical synthesis of N-acetyl enamides.

    Science.gov (United States)

    Tang, Wenjun; Capacci, Andrew; Sarvestani, Max; Wei, Xudong; Yee, Nathan K; Senanayake, Chris H

    2009-12-18

    A facile and practical method for the synthesis of N-acetyl alpha-arylenamides has been developed from corresponding ketoximes as the starting materials with ferrous acetate as the reducing reagent. This methodology offers mild reaction conditions, simple purification procedures, and high yields for a variety of N-acetyl enamides. PMID:19921804

  2. Unchanged acetylation of isoniazid by alcohol intake

    DEFF Research Database (Denmark)

    Wilcke, J T R; Døssing, M; Angelo, H R;

    2004-01-01

    no impact on the conversion of INH to its metabolite acetylisoniazid, which is catalysed by the enzyme N-acetyltranferase. Accordingly, a metabolic effect of acute alcohol intake on INH metabolism probably contributes little to the therapeutic failure of anti-tuberculosis treatment among alcoholics.......SETTING: In 10 healthy subjects, the influence of acute alcohol intake on the pharmacokinetics of isoniazid (INH) was studied. OBJECTIVE: To test the hypothesis that alcohol increases the conversion of INH by acetylation into its metabolite acetylisoniazid. DESIGN: In a crossover design, an oral...... dose of 300 mg INH was administered on 2 separate days, 14 days apart, with or without alcohol to a serum alcohol of about 21 mmol/l (1 g/l) maintained for 12 h. RESULTS: Neither the metabolism of INH nor that of acetylisoniazid was changed by acute alcohol intake. CONCLUSION: Acute alcohol intake has...

  3. Regulatory Light Chain Phosphorylation and N-Terminal Extension Increase Cross-Bridge Binding and Power Output in Drosophila at In Vivo Myofilament Lattice Spacing

    OpenAIRE

    Miller, Mark S.; Farman, Gerrie P.; Braddock, Joan M.; Soto-Adames, Felipe N.; Irving, Thomas C.; Vigoreaux, Jim O.; Maughan, David W.

    2011-01-01

    The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2+), truncated N-terminal extension (Dmlc2Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2S66A,S67A), and du...

  4. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Directory of Open Access Journals (Sweden)

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  5. Crystal structure of the rigor-like human non-muscle myosin-2 motor domain.

    Science.gov (United States)

    Münnich, Stefan; Pathan-Chhatbar, Salma; Manstein, Dietmar J

    2014-12-20

    We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8 Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50 kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A). PMID:25451231

  6. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    . The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...

  7. 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.

  8. Targeting a Dynamic Protein–Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex

    OpenAIRE

    Douse, Christopher H; Vrielink, Nina; Wenlin, Zhang; Cota, Ernesto; Tate, Edward W

    2014-01-01

    Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein–protein interaction represents an attractive...

  9. Pharmacological activation of myosin II paralogs to correct cell mechanics defects

    OpenAIRE

    Surcel, Alexandra; Ng, Win Pin; West-Foyle, Hoku; Zhu, Qingfeng; Ren, Yixin; Avery, Lindsay B.; Krenc, Agata K.; Meyers, David J.; Rock, Ronald S.; Anders, Robert A.; Freel Meyers, Caren L.; Robinson, Douglas N.

    2015-01-01

    Despite the integral role of cell mechanics, efforts to target mechanics for drug development have lagged. Here, we present an approach to identifying small molecules capable of modulating mechanics. We characterize 4-hydroxyacetophenone (4-HAP), isolated as a breakdown product of a hit from our pilot screen of over 22,000 compounds. We show that 4-HAP specifically alters the localization of the mechanoenzyme myosin II, increasing the stiffness of cells. The effect of 4-HAP on myosin II, whos...

  10. Nucleotide Dependent Intrinsic Fluorescence Changes of W29 and W36 in Smooth Muscle Myosin

    OpenAIRE

    van Duffelen, Marilyn; Chrin, Lynn R.; Berger, Christopher L.

    2004-01-01

    The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogen...

  11. 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.

  12. 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.

  13. Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model.

    Science.gov (United States)

    Skubiszak, Ludmila; Kowalczyk, Leszek

    2002-01-01

    Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created. PMID:12545190

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

    International Nuclear Information System (INIS)

    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

  15. 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.

  16. A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium.

    Science.gov (United States)

    Schwarz, E C; Geissler, H; Soldati, T

    1999-01-01

    In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins. PMID:10403059

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

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

    Science.gov (United States)

    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.

  20. Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine.

    Science.gov (United States)

    Mace, Oliver J; Morgan, Emma L; Affleck, Julie A; Lister, Norma; Kellett, George L

    2007-04-15

    Glucose absorption in rat jejunum involves Ca(2+)- and PKC betaII-dependent insertion of GLUT2 into the apical membrane. Ca(2+)-induced rearrangement of the enterocyte cytoskeleton is thought to enhance paracellular flow. We have therefore investigated the relationships between myosin II regulatory light chain phosphorylation (RLC(20)), absorption of glucose, water and calcium, and mannitol clearance. ML-7, an inhibitor of myosin light chain kinase, diminished the phloretin-sensitive apical GLUT2 but not the phloretin-insensitive SGLT1 component of glucose absorption in rat jejunum perfused with 75 mM glucose. Western blotting and immunocytochemistry revealed marked decreases in RLC(20) phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC betaII, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca(2+), or inhibition of unidirectional (45)Ca(2+) absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca(2+), nor clearance of [(14)C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with (45)Ca(2+) absorption or mannitol clearance. We conclude that the Ca(2+) necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Ca(v)1.3, and is dependent on SGLT1. Moreover, terminal web RLC(20) phosphorylation is necessary for apical GLUT2 insertion. The data confirm that glucose absorption by paracellular flow is negligible, and show further that paracellular flow makes no more than a minimal contribution to jejunal Ca(2+) absorption at luminal concentrations prevailing after a meal.

  1. Modelling the effect of myosin X motors on filopodia growth

    International Nuclear Information System (INIS)

    We present a numerical simulation study of the dynamics of filopodial growth in the presence of active transport by myosin X motors. We employ both a microscopic agent-based model, which captures the stochasticity of the growth process, and a continuum mean-field theory which neglects fluctuations. We show that in the absence of motors, filopodia growth is overestimated by the continuum mean-field theory. Thus fluctuations slow down the growth, especially when the protrusions are driven by a small number (10 or less) of F-actin fibres, and when the force opposing growth (coming from membrane elasticity) is large enough. We also show that, with typical parameter values for eukaryotic cells, motors are unlikely to provide an actin transport mechanism which enhances filopodial size significantly, unless the G-actin concentration within the filopodium greatly exceeds that of the cytosol bulk. We explain these observations in terms of order-of-magnitude estimates of diffusion-induced and advection-induced growth of a bundle of Brownian ratchets. (paper)

  2. Modelling the effect of myosin X motors on filopodia growth

    CERN Document Server

    Wolff, Katrin; Evans, Martin R; Goryachev, Andrew B; Marenduzzo, Davide

    2014-01-01

    We present a numerical simulation study of the dynamics of filopodial growth in the presence of active transport by myosin X motors. We employ both a microscopic agent-based model, which captures the stochasticity of the growth process, and a continuum mean-field theory which neglects fluctuations. We show that in the absence of motors, filopodia growth is overestimated by the continuum mean-field theory. Thus fluctuations slow down the growth, especially when the protrusions are driven by a small number (10 or less) of F-actin fibres, and when the force opposing growth (coming from membrane elasticity) is large enough. We also show that, with typical parameter values for eukaryotic cells, motors are unlikely to provide an actin transport mechanism which enhances filopodial size significantly, unless the G-actin concentration within the filopodium greatly exceeds that of the cytosol bulk. We explain these observations in terms of order-of-magnitude estimates of diffusion-induced and advection-induced growth o...

  3. Absence of platelet phenotype in mice lacking the motor protein myosin Va.

    Directory of Open Access Journals (Sweden)

    Matthew T Harper

    Full Text Available BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/- platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIbβ(3 activation, Ca(2+ signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/- platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.

  4. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    Science.gov (United States)

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased ...

  5. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J;

    2011-01-01

    -translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25......-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity...

  6. Function-structure relationships of acetylated pea starches

    OpenAIRE

    J. Huang

    2006-01-01

    Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different size fractions. The amount of introduced acetyl groups was found to depend on the size of the granules for the reaction with rapidly reacting reagent acetic acid anhydride, whereas the amount of intr...

  7. The growing landscape of lysine acetylation links metabolism and cell signalling

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya;

    2014-01-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, im...

  8. Hesperidin alleviates rat postoperative ileus through anti-inflammation and stimulation of Ca2+-dependent myosin phosphorylation

    Science.gov (United States)

    Xiong, Yong-jian; Chu, Hong-wei; Lin, Yuan; Han, Fang; Li, Ya-chan; Wang, Ai-guo; Wang, Fu-jin; Chen, Da-peng; Wang, Jing-yu

    2016-01-01

    Aim: Postoperative ileus (POI) is a postoperative dysmotility disorder of gastrointestinal tract, which remains one of the most perplexing problems in medicine. In the present study we investigated the effects of hesperidin, a major flavonoid in sweet oranges and lemons, on POI in rats. Methods: SD rats were administered hesperidin (5, 20, and 80 mg·kg−1·d−1, ig) for 3 consecutive days. POI operation (gently manipulating the cecum for 1 min) was performed on d 2. The gastrointestinal motility and isolated intestinal contraction were examined 1 d after the operation. Then the myosin phosphorylation and inflammatory responses in cecum tissue were assessed. Smooth muscle cells were isolated from rat small intestine for in vitro experiments. Results: The gastric emptying and intestinal transit were significantly decreased in POI rats, which were reversed by administration of hesperidin. In ileum and cecum preparations of POI rats in vitro, hesperidin (2.5–160 μmol/L) dose-dependently increased the spontaneous contraction amplitudes without affecting the contractile frequency, which was blocked by the myosin light chain kinase (MLCK) inhibitor ML-7 or verapamil, but not by TTX. Furthermore, administration of hesperidin increased the phosphorylation of MLC20 in the cecum tissue of POI rats. Moreover, administration of hesperidin reversed the increased levels of inflammatory cytokines, iNOS and COX-2 in cecum tissue of POI rats. In freshly isolated intestinal smooth muscle cells, hesperidin (5–80 μmol/L) dose-dependently increased the intracellular Ca2+ concentration as well as the phosphorylation of MLC20, which was abrogated by ML-7 or siRNA that knocked down MLCK. Conclusion: Oral administration of hesperidin effectively alleviates rat POI through inhibition of inflammatory responses and stimulation of Ca2+-dependent MLC phosphorylation. PMID:27345626

  9. Oil spill sorption using raw and acetylated sugarcane bagasse

    Institute of Scientific and Technical Information of China (English)

    Reza Behnood; Bagher Anvaripour; Nematollah Jaafarzadeh; Masoome Farasati

    2016-01-01

    In the recent decades oil spills in the aquatic environments are one of the major sources of environmental pollutions, which are steadily growing with the increase in oil consumption. Adsorption is a rapid and cost effective processto minimize the environmental impacts of oil spills andcleanup these pollutants. In this work, the crude oil sorption capacity was examined with raw sugarcane bagasse and acetylated sugarcane bagasse. Results show that the acetylated bagasse was significantly more oleophilic than the raw bagasse and acetylation reaction can increase bagasse oil sorption ability by about 90%. The maximum sorption capacities of acetylated bagasse were obtained about 11.3 g and 9.1 g in dry system (crude oil sorption) and oil layer sorption, respectively. The physicochemical characteristics of the sorbents such as composition, water solubility, moisture content and density were measured according to ASTM standard methods. Also Fourier transform infrared spectroscopy (FTIR) of raw and acetylated bagasse was performed to investigate the effect of acetylation on sugarcane bagasse structure.

  10. Site-specific acetylation of ISWI by GCN5

    Directory of Open Access Journals (Sweden)

    Chioda Mariacristina

    2007-08-01

    Full Text Available Abstract Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(KacxPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

  11. Acetyl radical generation in cigarette smoke: Quantification and simulations

    Science.gov (United States)

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  12. Axonal transport and incorporation of radioactivity after injection of N-[3H]acetyl-D-mannosamine into rat mesencephalon

    International Nuclear Information System (INIS)

    A study has been performed to demonstrate the possibility of incorporation of sialic acid into nerve endings of the rubrospinal tract after antegrade axonal transport. Young adult rats received injections of N-[3H]acetyl-D-mannosamine into the red nucleus and axonal transport of the tritiated compounds along the axons of afferent and efferent connections of the red nucleus was studied and the transported material was analysed. Light microscopic autoradiography and biochemical methods were used. (Auth./C.F.)

  13. Hydrogen sulfide donor NaHS induces death of alveolar epithelial L2 cells that is associated with cellular shrinkage, transgelin expression and myosin phosphorylation.

    Science.gov (United States)

    Fujii, Yusuke; Funakoshi, Takeshi; Unuma, Kana; Noritake, Kanako; Aki, Toshihiko; Uemura, Koichi

    2016-01-01

    Hydrogen sulfide (H2S) is a highly toxic gaseous molecule that causes death to humans exposed to high concentrations. H2S is absorbed into the body through the alveolar epithelium and other tissues. The aim of this study is to evaluate the molecular mechanism underling acute lung injury caused by the inhalation of high concentrations of H2S. Rat lung epithelium-derived L2 cells were exposed to a H2S donor, NaHS, at concentrations of 2-4 mM for 1-6 hr. NaHS caused shrinkage and death of the cells without caspase activation. An actin-binding protein, transgelin, was identified as one of the NaHS-inducible proteins in the cells. NaHS increased myosin light chain (MLC) phosphorylation, indicating that actomyosin-mediated cellular contractility and/or motility could be increased after NaHS exposure. The administration of ML-7, a myosin light chain kinase (MLCK) inhibitor, accelerated cell death after NaHS exposure. Based on these data, we conclude that the increase in MLC phosphorylation in response to NaHS exposure is a cellular protective reaction against NaHS toxicity. Enhancements in smooth muscle cell properties such as transgelin expression and actomyosin-mediated contractility/motility might be involved in cell survival after NaHS exposure. PMID:27665774

  14. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

    Science.gov (United States)

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas; Houdusse, Anne; Kerkhoff, Eugen

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. PMID:27623148

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. 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.

  1. Stability of carboplatin, paclitaxel, and docetaxel with acetyl-l-carnitine during simulated Y-site administration.

    Science.gov (United States)

    Zhang, Yang; Scarlett, Cameron; Hutson, Paul

    2012-01-01

    The compatibility of acetyl-l-carnitine with three chemotherapy agents was studied during simulated Y-site administration. Acetyl-l-carnitine 30 mg/mL in 5% dextrose for injection (D5W) was combined with carboplatin 4 mg/mL, paclitaxel 2 mg/mL, and docetaxel 0.74 mg/mL in glass vials. Physical compatibility over the 4-hour storage at room temperature was assessed by visual examinations with unaided eye under fluorescent light and by measuring the percent transmittance at 600 nm. Chemical compatibility was measured by the percent of initial concentration remaining using stability-indicating high-performance liquid chromatographic-ultraviolet and high-performance liquid chromatographic-mass spectrometry methods. No visible particulate matter, haze, or color change was observed, and the percent transmittance was >95% for all admixtures. After a 4-hour incubation, 93.2% of the paclitaxel and 96.5% of docetaxel remained in separate mixtures with acetyl-l-carnitine. Carboplatin 4 mg/mL, paclitaxel 1.2 mg/mL, and docetaxel 0.74 mg/mL are physically and chemically compatible with acetyl-l-carnitine 30 mg/mLin D5W during a simulated 4-hour Y-site administration. PMID:23050317

  2. A dysregulated acetyl/SUMO switch of FXR promotes hepatic inflammation in obesity

    OpenAIRE

    Kim, Dong-Hyun; Xiao, Zhen; Kwon, Sanghoon; Sun, Xiaoxiao; Ryerson, Daniel; Tkac, David; Ma, Ping; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Zhou, Edward; Xu, H. Eric; Palvimo, Jorma J; Chen, Lin-Feng; Kemper, Byron; Kemper, Jongsook Kim

    2014-01-01

    Acetylation of transcriptional regulators is normally dynamically regulated by nutrient status but is often persistently elevated in nutrient-excessive obesity conditions. We investigated the functional consequences of such aberrantly elevated acetylation of the nuclear receptor FXR as a model. Proteomic studies identified K217 as the FXR acetylation site in diet-induced obese mice. In vivo studies utilizing acetylation-mimic and acetylation-defective K217 mutants and gene expression profilin...

  3. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    Directory of Open Access Journals (Sweden)

    Malin Persson

    Full Text Available Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached" or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  4. 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 he...

  5. Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.

    Science.gov (United States)

    Forer, A; Spurck, T; Pickett-Heaps, J D

    2007-01-01

    We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. PMID:18094930

  6. Inverse interaction between tropomyosin and phosphorylated myosin in the presence or absence of caldesmon

    Institute of Scientific and Technical Information of China (English)

    Ying Zhang; Houli Zhang; Zeyao Tang; Kazuhiro Kohama; Yuan Lin

    2013-01-01

    In the present study,co-sedimentation assay,intrinsic fluorescence intensity measurement,and Mg2+-ATPase activity analysis were carried out to investigate the direct effect of tropomyosin (TM) on unphosphorylated myosin (UM) or phosphorylated myosin (PM) in the presence or absence of caldesmon (CaD).Results showed that TM significantly decreased the sedimentation,intrinsic fluorescence intensity,and the Mg2+-ATPase activity of PM,but not UM.In the presence of CaD,TM also significantly decreased these parameters irrespective of myosin phosphorylation,suggesting that the interaction between TM and CaD abolished the effects of TM on PM or UM and that there was an inverse interaction between TM and PM,characterized by the decreased PM sedimentation and intrinsic fluorescence intensity.

  7. Novel Dictyostelium unconventional myosin, MyoM, has a putative RhoGEF domain.

    Science.gov (United States)

    Oishi, N; Adachi, H; Sutoh, K

    2000-05-26

    We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM(-) mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis. PMID:10828443

  8. Twirling motion of actin filaments in gliding assays with non-processive myosin motors

    CERN Document Server

    Vilfan, Andrej

    2009-01-01

    We present a model study of gliding assays in which actin filaments are moved by non-processive myosin motors. We show that even if the power stroke of the motor protein has no lateral component, the filaments will rotate around their axis while moving over the surface. Notably, the handedness of this twirling motion is opposite from that of the actin filament structure. It stems from the fact that the gliding actin filament has "target zones" where its subunits point towards the surface and are therefore more accessible for myosin heads. Each myosin head has a higher binding probability before it reaches the center of the target zone than afterwards, which results in a left-handed twirling. We present a stochastic simulation and an approximative analytical solution. The calculated pitch of the twirling motion depends on the filament velocity (ATP concentration). It reaches about 400nm for low speeds and increases with higher speeds.

  9. 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.

  10. Preparation of monoclonal antibodies against cardiac myosin and some radiolabelling studies

    International Nuclear Information System (INIS)

    Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125I and 99mTc. Human cardiac myosin was isolated from normal human heart and was used for raising the monoclonal antibodies by the hybridoma technique. Antibody producing clones were identified by ELISA and cloning was done by the limiting dilution technique. Of the 13 clones obtained, 4 were deemed suitable for further studies. The antibodies were grown in ascites, purified, isotyped and their cross reactions with other forms of myosin were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted to different extents with bovine skeletal myosin. The most avid antibody Mab-4G4 was chosen for further labelling studies. Mab-4G4 was labelled with 125I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. Purified radioiodinated antibody with radiochemical purity >95% could be obtained by gel filtration. Immunoreactivity was retained as tested by binding to myosin immobilised on a solid support. Mab-4G4 was also labelled with 99mTc using stannous tartrate as the reducing agent. Radiolabelling yield was ∼60%, the purity was >95% and the immunoreactivity was retained. Both the labelled preparations were tested for bio-distribution in normal and infarcted rats. The activity accumulation in the infarcted region was ∼ 1.5 and 3.5 times as that in normal heart muscle for 125I and 99mTc labelled Mab-4G4 respectively. The major problem with the iodinated antibody was the in vivo deiodination resulting in very high percentage of activity in the thyroid. Although the fraction of the total activity associated with the infarcted heart is not very impressive, the fact that the activities with the infarcted and normal hearths are significantly different is heartening. With further optimisation of labelling and use of F(ab)'2 fragments, better delineation of the infarct sites is aspired. (author)

  11. 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. PMID:26426395

  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. 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.

  14. 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

  15. Radiation-induced myosin IIA expression stimulates collagen type I matrix reorganization

    International Nuclear Information System (INIS)

    Background and purpose: Extracellular matrix (ECM) reorganization critically contributes to breast cancer (BC) progression and radiotherapy response. We investigated the molecular background and functional consequences of collagen type I (col-I) reorganization by irradiated breast cancer cells (BCC). Materials and methods: Radiation-induced (RI) col-I reorganization was evaluated for MCF-7/6, MCF-7/AZ, T47D and SK-BR-3 BCC. Phase-contrast microscopy and a stressed matrix contraction assay were used for visualization and quantification of col-I reorganization. Cell–matrix interactions were assessed by the inhibition of β1 integrin (neutralizing antibody ‘P5D2’) or focal adhesion kinase (FAK; GSK22560098 small molecule kinase inhibitor). The role of the actomyosin cytoskeleton was explored by western blotting analysis of myosin II expression and activity; and by gene silencing of myosin IIA and pharmacological inhibition of the actomyosin system (blebbistatin, cytochalasin D). BCC death was evaluated by propidium iodide staining. Results: We observed a radiation dose-dependent increase of col-I reorganization by BCC. β1 Integrin/FAK-mediated cell–matrix interactions are essential for RI col-I reorganization. Irradiated BCC are characterized by increased myosin IIA expression and myosin IIA-dependent col-I reorganization. Moreover, RI col-I reorganization by BCC is associated with decreased BCC death, as suggested by pharmacological targeting of the β1 integrin/FAK/myosin IIA pathway. Conclusions: Our data indicate the role of myosin IIA in col-I reorganization by irradiated BCC and reciprocal BCC death

  16. Opening the Arg-Glu salt bridge in myosin: computational study.

    Science.gov (United States)

    Kaliman, Ilya; Grigorenko, Bella; Shadrina, Maria; Nemukhin, Alexander

    2009-06-28

    Opening the Arg-Glu salt bridge in myosin, which presumably succeeds the myosin-catalyzed hydrolysis of adenosine triphosphate, was modeled computationally on the basis of the structures corresponding to the enzyme-substrate and enzyme-product complexes found in the quantum mechanics-molecular mechanics simulations. According to the calculations of the potential of mean force, opening the bridge is considerably facilitated upon termination of the chemical reaction, but does not promote egress of inorganic phosphate by the back-door mechanism. PMID:19506754

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

    OpenAIRE

    Katarzyna A. Makowska; Ruth E. Hughes; Kathryn J. White; Claire M. Wells; Michelle Peckham

    2015-01-01

    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-mus...

  18. SLOW MYOSIN ATP TURNOVER IN THE SUPER-RELAXED STATE IN TARANTULA MUSCLE

    OpenAIRE

    Naber, Nariman; Cooke, Roger; Pate, Edward

    2011-01-01

    We measured the nucleotide turnover rate of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ~30 minutes. This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated...

  19. Curcumin-induced Histone Acetylation in Malignant Hematologic Cells

    Institute of Scientific and Technical Information of China (English)

    Junbin HU; Yan WANG; Yan CHEN

    2009-01-01

    This study investigated the inhibitory effects of curcumin on proliferation of hemato-logical malignant cells in vitro and the anti-tumor mechanism at histone acetylation/histone deacety-lation levels.The effects of curcumin and histone deacetylase inhibitor trichostatin A (TSA) on the growth of Raji cells were tested by MTT assay.The expression of acetylated histone-3 (H3) in Raji,HL60 and K562 cells,and peripheral blood mononuclear cells (PBMCs) treated with curcumin or TSA was detected by immunohistochemistry and FACS.The results showed curcumin inhibited pro-liferation of Raji cells significantly in a time- and dose-dependent fashion,while exhibited low toxic-ity in PBMCs.Curcumin induced up-regulation of the expression of acetylated H3 dose-dependently in all malignant cell lines tested.In conclusion,curcumin inhibited proliferation of Raji cells selec-tively,enhanced the level of acetylated H3 in Raji,HL60,and K562 cells,which acted as a histone deacetylase inhibitor like TSA.Furthermore,up-regulation of H3 acetylation may play an important role in regulating the proliferation of Raji cells.

  20. Recycling and Reuse of Ionic Liquid in Homogeneous Cellulose Acetylation

    Institute of Scientific and Technical Information of China (English)

    HUANG Kelin; WU Rui; CAO Yan; LI Huiquan; WANG Jinshu

    2013-01-01

    Molecular distillation was used to recover ionic liquid (IL) 1-allyl-3-methylimidazolium chloride (AmimC1) in homogeneous cellulose acetylation.The five factors that affect the separation efficiency of molecular distillation,namely,feed flow rate,distillation temperature,feed temperature,wiper rotating speed,and distillation pressure,are discussed.The optimal recovery condition was determined via orthogonal experiments using an OA9(34) design.The IL was recycled and reused 5 times in the homogeneous cellulose acetylation system under optimal conditions.The purity of recycled IL the 5th time reached 99.56%.FT-IR (Fourier transform infrared spectroscopy) and 1H NMR (nuclear magnetic resonance) spectroscopy showed that the structure of the recovered IL is not changed.This work proves that AmirnCl has excellent reusability,and that molecular distillation is an effective method for recovering IL in homogeneous cellulose acetylation.

  1. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis

    Directory of Open Access Journals (Sweden)

    Jialei Hu

    2015-12-01

    Full Text Available Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination.

  2. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    Science.gov (United States)

    Ristić, I. S.; Nikolić, L.; Nikolić, V.; Ilić, D.; Budinski-Simendić, J.

    2011-12-01

    Polyrotaxanes are intermediary products in the synthesis of topological gels. They are created by inclusion complex formation of hydrophobic linear macromolecules with cyclodextrins or their derivatives. Then, pairs of cyclodextrin molecules with covalently linkage were practically forming the nodes of the semi-flexible polymer network. Such gels are called topological gels and they can absorb huge quantities of water due to the net flexibility allowing the poly(ethylene oxide) chains to slide through the cyclodextrin cavities, without being pulled out altogether. For polyrotaxane formation poly(ethylene oxide) was used like linear macromolecules. There are hydroxyl groups at poly(ethylene oxide) chains, whereby the linking of the voluminous molecules should be made. To avoid the reaction of cyclodextrin OH groups with stoppers, they should be protected by, e.g., acetylation. In this work, the acetylation of the OH groups of β-cyclodextrin was performed by acetic acid anhydride with iodine as the catalyst. The acetylation reaction was assessed by the FTIR and HPLC method. By the HPLC analysis was found that the acetylation was completed in 20 minutes. Inserting of poly(ethylene oxide) with 4000 g/mol molecule mass into acetyl-β-cyclodextrin with 2:1 poly(ethylene oxide) monomer unit to acetyl-β-cyclodextrin ratio was also monitored by FTIR, and it was found that the process was completed in 12 h at the temperature of 10°C. If the process is performed at temperatures above 10°C, or for periods longer than 12 hours, the process of uncontrolled hydrolysis of acetate groups was initiated.

  3. Remote control of molecular motors using light-activated gearshifting

    Science.gov (United States)

    Bryant, Zev

    2013-03-01

    Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in vivo and provide sophisticated components for directed nanoscale transport in vitro. We previously constructed myosin motors that respond to a change in [Ca++] by reversing their direction of motion along the polarized actin filament. To expand the potential applications of controllable molecular motors, we have now developed myosins that shift gears in response to blue light illumination. Light is a versatile control signal that can be readily modulated in time and space, and is generally orthogonal to cellular signaling. Using structure-guided protein engineering, we have incorporated LOV photoreceptor domains into the lever arms of chimeric myosins, resulting in motors that robustly speed up, slow down, or switch directions upon illumination. These genetically encoded motors should be directly deployable inside living cells. Our successful designs include constructs based on two different myosin classes, and we show that optical velocity control can be implemented in motors that move at microns/sec speeds, enabling practical biological and bioengineering applications.

  4. Study on Reactions of 2-Acetyl-7-methylaminotropone with Pyridinecarboxyaldehydes

    Institute of Scientific and Technical Information of China (English)

    GAO Wen-Tao; ZHENG Zhuo

    2003-01-01

    @@ Cinnamoyl group is a versatile constituent because of bearing active carbonyl group and α, β-unsaturated car bon-carbon double bond. A wide variety of heterocycle-fused troponoid compounds have been derived from cinnamoyl-substituted tropones. For examples, the 3-(4-aryl-3-cyano-2- methoxypyridin-6-yl)tropones were synthesized by the reactions of 2-acetyl-7-methylaminotropone with malononitrile via michael addition and cyclization. [1] There have been some reports about the synthesis of 2-cinnamoyl-7-methylaminotropone, [2,3] herein we further report the synthesis of this kind of compounds by the reactions of 2-acetyl-7-methylaminotropone with pyridinecarboxyaldehydes.

  5. Model of myosin recruitment to the cell equator for cytokinesis: feedback mechanisms and dynamical regimes

    Science.gov (United States)

    Veksler, Alexander; Vavylonis, Dimitrios

    2011-03-01

    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During animal cell cytokinesis, cortical myosin filaments (MF) disassemble at the flanking regions and concentrate in the equator. This recruitment depends on myosin motor activity and the Rho proteins that regulate MF assembly and disassembly. Central spindle and astral microtubules help establish a spatial pattern of differential Rho activity. We propose a reaction-diffusion model for the dynamics of MF recruitment to the equatorial region. In the model, the central spindle and mechanical stress promote self-reinforcing MF assembly. Negative feedback is introduced by MF-induced recruitment of inhibitor myosin phosphatase. Our model yields various dynamical regimes and explains both the recruitment of MF to the cleavage furrow and the observed damped MF oscillations in the flanking regions, as well as steady MF assembly. Space and time parameters of MF oscillations are calculated. We predict oscillatory relaxation of cortical MF upon removal of locally-applied external stress.

  6. 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.

  7. Comparison of the variable loop regions of myosin heavy chain genes from Antarctic and temperate isopods.

    Science.gov (United States)

    Holmes, J M; Whiteley, N M; Magnay, J L; El Haj, A J

    2002-03-01

    The evolutionary adaptations of functional genes to life at low temperatures are not well characterised in marine and fresh water invertebrates. Temperature has been shown to affect the functional characteristics of fish muscles, with changes in the velocity of shortening and ATPase activity being associated with myosin heavy chain (MyHC) isoform composition and the structure of the surface loop regions. Two PCR products spanning loops 1 and 2 of a MyHC gene from an Antarctic isopod (Glyptonotus antarcticus) were sequenced and compared with those of a temperate isopod (Idotea resecata), slow and fast fibres from lobster (Homarus gammarus) and a cold water amphipod (Eulimnogammarus verrucosus), revealing specific differences between the species, possibly related to fibre type and habitat temperature. The loop 2 region from G. antarcticus myosin was cloned and used for Northern analysis of total RNA from the other species. The cloned myosin cDNA hybridised specifically to a 6.6-kb transcript, in G. antarcticus muscle. In contrast, cDNA probes for lobster slow myosin and actin hybridised to muscle RNA from all species, demonstrating that a distinct MyHC isoform is expressed in the Antarctic isopod, as opposed to the temperate species. The inter- and intra-specific sequence differences in loop 2 region suggest that this may be a site for muscle adaptation to enable function at the low temperatures found in the Southern Ocean. PMID:11959017

  8. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    Energy Technology Data Exchange (ETDEWEB)

    Schoenitzer, Veronika [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Eichner, Norbert [Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Clausen-Schaumann, Hauke [Munich University of Applied Sciences, Lothstrasse 34, D-80335 Muenchen, Germany, and Center for NanoScience (CeNS), Geschwister-Scholl-Platz 1, D-80539 Muenchen (Germany); Weiss, Ingrid M., E-mail: ingrid.weiss@inm-gmbh.de [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  9. 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.

  10. Study on the tripolyphosphatase (TPPase) property of bighead carp ( Aristichthys nobilis) myosin subfragment-1

    Science.gov (United States)

    Gao, Ruichang; Xue, Changhu; Yuan, Li; Zhang, Yongqin; Xue, Yong; Sun, Yan; Feng, Hui

    2007-10-01

    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 30°C 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 0.3 molL-1 under the condition of 17 mmolL-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 K M of 3.2mmolL-1.

  11. 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.

  12. 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.

  13. Brush border myosin Ia inactivation in gastric but not endometrial tumors

    NARCIS (Netherlands)

    Mazzolini, Rocco; Rodrigues, Paulo; Bazzocco, Sarah; Dopeso, Higinio; Ferreira, Ana M.; Mateo-Lozano, Silvia; Andretta, Elena; Woerner, Stefan M.; Alazzouzi, Hafid; Landolfi, Stefania; Hernandez-Losa, Javier; Macaya, Irati; Suzuki, Hiromu; Ramon y Cajal, Santiago; Mooseker, Mark S.; Mariadason, John M.; Gebert, Johannes; Hofstra, Robert M. W.; Reventos, Jaume; Yamamoto, Hiroyuki; Schwartz, Simo; Arango, Diego

    2013-01-01

    Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MS

  14. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    Directory of Open Access Journals (Sweden)

    Shuji Ueda

    Full Text Available Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.

  15. 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.

  16. 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. PMID:26593463

  17. New lysine-acetylated proteins screened by immunoaffinity and liquid chromatography-mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The lack of selective extraction specific for lysine-acetylated proteins has been a major problem in the field of acetylation biology,though acetylation plays a key role in many biological processes.In this paper,we report for the first time the proteomic screening of lysine-acetylated proteins from a mouse liver tissue,by a new approach of immunoaffinity purification of lysine-acetylated peptides combined with nano-HPLC/MS/MS analysis.We have found 20 lysine-acetylated proteins with 21 lysine-acetylated sites,among which 12 lysine-acetylated proteins and 16 lysine-acetylated sites have never been reported before.Notably,three acetyltransferases harboring in mitochondrion are newly discovered acetyltransferases responsible for the acetylation of nonhistone proteins.We have explored the significant patterns of residue preference by the hierarchical clustering analysis of amino acid residues surrounding acetylation sites,which could be helpful to the prediction of new sites of lysine acetylation.Our findings provide more candidates for studying the important roles played by acetylation in diverse cellular pathways and related human diseases.

  18. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation.

    Science.gov (United States)

    Homburger, Julian R; Green, Eric M; Caleshu, Colleen; Sunitha, Margaret S; Taylor, Rebecca E; Ruppel, Kathleen M; Metpally, Raghu Prasad Rao; Colan, Steven D; Michels, Michelle; Day, Sharlene M; Olivotto, Iacopo; Bustamante, Carlos D; Dewey, Frederick E; Ho, Carolyn Y; Spudich, James A; Ashley, Euan A

    2016-06-14

    Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

  19. 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.

  20. 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.

  1. Harmonic Force Spectroscopy Reveals a Force-Velocity Curve from a Single Human Beta Cardiac Myosin Motor

    DEFF Research Database (Denmark)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian L.;

    2014-01-01

    human beta cardiac myosin S1. We also compare load-velocity curves for wild-type motors with load-velocity curves of mutant forms that cause hypertrophic or dilated-cardiomyopathy (HCM or DCM), in order to understand the effects of mutations on the contractile cycle at the single molecule level....... is slow under high load and fast under low load. We use a new, simple method we call "harmonic force spectroscopy" to extract a load-velocity relationship from a single human beta cardiac myosin II motor (S1). With a dual-beam optical trap, we hold an actin dumbbell over a single myosin molecule...

  2. SCANDIUM TRIFLATE CATALYZED ACETYLATION OF STARCH UNDER MILD CONDITIONS

    Science.gov (United States)

    Scandium (III) trifluoromethan sulfonate (Sc(OTf)3) was investigated as a catalyst for the acetylation of starch in order to determine the potential for preparing new types of starch esters under mild conditions. At room temperature, dry granular corn starch reacts with acetic anhydride in the pres...

  3. The potential role of wood acetylation in climate change mitigation

    NARCIS (Netherlands)

    Van der Lugt, P.; Vogtländer, J.G.; Alexander, J.; Bongers, F.; Stebbins, H.

    2014-01-01

    In a carbon footprint assessment, the greenhouse gas emissions during the life cycle of a material can be measured, and compared to alternative products in terms of kg CO2 equivalent. If applied correctly, wood acetylation opens up a range of new innovative applications in which high performance yet

  4. Surface effects in the acetylation of granular potato starch

    NARCIS (Netherlands)

    Steeneken, P.A.M.; Woortman, A.J.J.

    2008-01-01

    The occurrence of surface effects in the acetylation of granular potato starch with acetic anhydride to degrees of substitution 0.04-0.2 was studied by two different approaches. The first approach involved the fractionation of granular starch acetates into five different size classes and analysis of

  5. Predicting post-translational lysine acetylation using support vector machines

    DEFF Research Database (Denmark)

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram;

    2010-01-01

    spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  6. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    Science.gov (United States)

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  7. Function-structure relationships of acetylated pea starches

    NARCIS (Netherlands)

    Huang, J.

    2006-01-01

    Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different si

  8. Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome.

    Science.gov (United States)

    Kim, Jongseong; Lee, Jaehyoun; Lee, Tae-Hee

    2015-12-01

    The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit.

  9. Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response.

    Science.gov (United States)

    Ghari, Fatemeh; Quirke, Anne-Marie; Munro, Shonagh; Kawalkowska, Joanna; Picaud, Sarah; McGouran, Joanna; Subramanian, Venkataraman; Muth, Aaron; Williams, Richard; Kessler, Benedikt; Thompson, Paul R; Fillipakopoulos, Panagis; Knapp, Stefan; Venables, Patrick J; La Thangue, Nicholas B

    2016-02-01

    Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression. PMID:26989780

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    Lifescience Database Archive (English)

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  12. File list: His.Oth.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Kid.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: His.Myo.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: His.Lng.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: His.Pan.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.Unc.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.CDV.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.Dig.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Unc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Prs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  4. File list: His.Utr.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.Unc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Oth.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.Adp.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Gon.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.Dig.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.Adp.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  15. File list: His.Myo.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: His.Brs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Oth.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

  4. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  5. Histone H3 acetylation in the postmortem Parkinson's disease primary motor cortex.

    Science.gov (United States)

    Gebremedhin, Kibrom G; Rademacher, David J

    2016-08-01

    Although the role of epigenetics in Parkinson's disease (PD) has not been extensively studied, α-synuclein, the main component of Lewy bodies, decreased histone H3 acetylation. Here, we determined if there were histone acetylation changes in the primary motor cortex which, according to the Braak model, is one of the last brain regions affected in PD. Net histone H3 acetylation, histone H3 lysine 9 (H3K9), histone H3 lysine 14 (H3K14), histone H3 lysine 18 (H3K18), and histone H3 lysine 23 (H3K23) acetylation was assessed in the primary motor cortex of those affected and unaffected by PD. There was net increase in histone H3 acetylation due to increased H3K14 and H3K18 acetylation. There was a decrease in H3K9 acetylation. No between-groups difference was detected in H3K23 acetylation. Relationships between Unified Lewy Body Staging scores and histone H3 acetylation and substantia nigra depigmentation scores and histone H3 acetylation were observed. No relationships were detected between postmortem interval and histone H3 acetylation and expired age and histone H3 acetylation. These correlational data support the notion that the histone H3 acetylation changes observed here are not due to the postmortem interval or aging. Instead, they are due to PD and/or factors that covary with PD. The data suggest enhanced gene transcription in the primary motor cortex of the PD brain due to increase H3K14 and H3K18 acetylation. This effect is partially offset by a decreased H3K9 acetylation, which might repress gene transcription. PMID:27241718

  6. Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro.

    OpenAIRE

    Chaen, S; Oiwa, K; Shimmen, T; Iwamoto, H; Sugi, H

    1989-01-01

    To elucidate the molecular mechanism of muscle contraction resulting from the ATP-dependent actin-myosin interaction, we constructed an assay system with which both the force and the movement produced by the actin-myosin interaction in vitro can be simultaneously recorded and analyzed. The assay system consisted of the giant internodal cells of an alga, Nitellopsis obtusa, which contain well-organized arrays of actin filaments (actin cables) running along the cell long axis, and a glass micro...

  7. 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.

  8. Tubule-guided cell-to-cell movement of a plant virus requires class XI myosin motors.

    Directory of Open Access Journals (Sweden)

    Khalid Amari

    2011-10-01

    Full Text Available Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD, organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.

  9. ACETYLATION INCREASES EWS-FLI1 DNA BINDING AND TRANSCRIPTIONAL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Silke eSchlottmann

    2012-09-01

    Full Text Available Ewing Sarcoma (ES is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant posttranslational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors, and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with histone deacetylase inhibitors (HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in COS7 cells. However, our data that evaluates the acetylation of ful-length EWS-FLI1 remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  10. A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

    Science.gov (United States)

    Li, G; Sanchez, V; Nagaraj, P C S B; Khan, S; Rajpoot, N

    2015-12-01

    We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two-motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state-of-the-art tracking methods, especially for sequences with high spot density. PMID:26259144

  11. 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.

  12. 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.

  13. N-Acetyltransferase 2 (NAT2) in Tunisian Population: Correlation Between Acetylation Phenotype and Genotype

    International Nuclear Information System (INIS)

    One hundred tuberculous patients were studied during 2004-2005 to determine acetylation phenotype, frequent mutations of NAT2 gene and to compare acetylation phenotype with NAT2 genotype in Tunisian population. Acetylation phenotype was determined by determination of acetylation index. Five mutations of NAT2 gene were evaluated by PCR/RFLP. Results show bimodal distribution of acetylation SA and RA phenotype, 75% and 25% and genotype 56% and 44%, respectively. Ten NAT2 alleles were found, NAT2*4 being the major one. Thirty-two different genotypes were found (9 RA and 23 SA). The major one was NAT2*6 B/NAT2*4. The concordance value was 79%. A good sensibility (98, 2%) of acetylation test for SA detection was found. Thus, acetylation phenotype in SA is predicted with poor error risk. (author)

  14. Histone acetylation dependent energy landscapes in tri-nucleosome revealed by residue-resolved molecular simulations

    Science.gov (United States)

    Chang, Le; Takada, Shoji

    2016-01-01

    Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366

  15. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches

    OpenAIRE

    Velten, Brandy P.; Welch, Kenneth C.

    2014-01-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25–60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to...

  16. 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.

  17. Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.

    Science.gov (United States)

    Spiró, Zoltán; Thyagarajan, Kalyani; De Simone, Alessandro; Träger, Sylvain; Afshar, Katayoun; Gönczy, Pierre

    2014-07-01

    Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension. PMID:24961801

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

    OpenAIRE

    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...

  19. Expression and DNA sequence analysis of a human embryonic skeletal muscle myosin heavy chain gene.

    OpenAIRE

    Karsch-Mizrachi, I; M. Travis; Blau, H; Leinwand, L A

    1989-01-01

    Vertebrate myosin heavy chains (MHC) are represented by multiple genes that are expressed in a spatially and temporally distinct pattern during development. In order to obtain molecular probes for developmentally regulated human MHC isoforms, we used monoclonal antibodies to screen an expression cDNA library constructed from primary human myotube cultures. A 3.4 kb cDNA was isolated that encodes one of the first MHCs to be transcribed in human skeletal muscle development. A portion of the cor...

  20. Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

    OpenAIRE

    Trus, B L; Steven, A C; McDowall, A W; M. Unser; Dubochet, J; Podolsky, R J

    1989-01-01

    For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In ...

  1. 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...

  2. The SAH domain extends the functional length of the myosin lever.

    Science.gov (United States)

    Baboolal, Thomas G; Sakamoto, Takeshi; Forgacs, Eva; White, Howard D; Jackson, Scott M; Takagi, Yasuharu; Farrow, Rachel E; Molloy, Justin E; Knight, Peter J; Sellers, James R; Peckham, Michelle

    2009-12-29

    Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins. PMID:20018767

  3. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics

    OpenAIRE

    Ching-Wei Chang; Sanjay Kumar

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual ...

  4. Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB

    OpenAIRE

    Rolo, Ana; Skoglund, Paul; Keller, Ray

    2008-01-01

    Vertebrate neural tube formation involves two distinct morphogenetic events -convergent extension (CE) driven by medio-lateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation ...

  5. The potential role of wood acetylation in climate change mitigation

    OpenAIRE

    Van der Lugt, P.; Vogtländer, J.G.; J. Alexander; Bongers, F.; Stebbins, H.

    2014-01-01

    In a carbon footprint assessment, the greenhouse gas emissions during the life cycle of a material can be measured, and compared to alternative products in terms of kg CO2 equivalent. If applied correctly, wood acetylation opens up a range of new innovative applications in which high performance yet carbon intensive non-renewable materials such as metals, plastics and concrete may be replaced by abundantly available nondurable wood species. To better understand the difference in greenhouse ga...

  6. 4-Acetyl-1H-pyrrole-2-carbaldehyde

    Directory of Open Access Journals (Sweden)

    Tao Sun

    2012-10-01

    Full Text Available The title compound, C7H7NO2, was synthesized via a one-pot Vilsmeier–Haack and subsequent Friedel–Crafts reaction. The pyrazole ring makes dihedral angles of 4.50 (9 and 2.06 (8°, respectively, with the aldehyde and acetyl groups. In the crystal, classical N—H...O hydrogen bonds and weak C—H...O interactions assemble the molecules into a chain along the b axis.

  7. A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells

    Directory of Open Access Journals (Sweden)

    Justyna Karolczak

    2015-01-01

    Full Text Available Myosin VI (MVI is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9, a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.

  8. A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells.

    Science.gov (United States)

    Karolczak, Justyna; Sobczak, Magdalena; Skowronek, Krzysztof; Rędowicz, Maria Jolanta

    2015-01-01

    Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.

  9. Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

    Science.gov (United States)

    Zawadowska, B; Majerczak, J; Semik, D; Karasinski, J; Kolodziejski, L; Kilarski, W M; Duda, K; Zoladz, J A

    2004-01-01

    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 (Pmodern 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. PMID:15493580

  10. 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.

  11. 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.

  12. Myosin heavy chain-based fibre types in red cell hyper- and normovolaemic Standardbred trotters.

    Science.gov (United States)

    Karlström, K; Essén-Gustavsson, B

    2002-09-01

    An assumed link between red cell hypervolaemia, an excessive amount of training and impaired performance of hypervolaemic horses has led to a theory that the muscle fibres could be affected. Myosin heavy chain (MHC)-based fibre type composition in gluteus medius muscle of red blood cell normo- (NV) and hypervolaemic (HV) Standardbred trotters was evaluated using immunohistochemistry. Muscle biopsies were obtained from 13 NV and 16 HV horses. Serial transverse sections were cut and reacted with antibodies against different isoforms of the myosin heavy chains MHCI, MHCIIA and MHCIIX. Sections were also stained for myofibrillar ATPase pH 4,6 to identify types I, IIA and IIB, and NADH tetrazolium reductase to evaluate the oxidative capacity. The results show that types I and IIA fibres corresponded between staining methods, whereas IIB fibres in the ATPase stains were more numerous than pure MHCIIX fibres from immunohistochemistry. Many fibres identified histochemically as type IIB fibres contained both MHC isoforms IIA and IIX (MHCIIAX). Most fibres had a high oxidative capacity, but among the fibres within a section, the lowest was seen subjectively in pure MHCIIX fibres. Immunohistochemical stains make it possible to detect differences in fibre type composition that are not observed with myosin ATPase stainings, as it was found that HV horses had a lower percentage of MHCIIX fibres than NV horses. Immunohistochemical methods are, therefore, valuable for use in further research and clinical studies concerning muscle adaptations. PMID:12405701

  13. 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

    -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...... 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...

  14. Acetylation modification regulates GRP78 secretion in colon cancer cells.

    Science.gov (United States)

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  15. Acetylation modification regulates GRP78 secretion in colon cancer cells

    Science.gov (United States)

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  16. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    Science.gov (United States)

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p processes. We have shown aberrant histone acetylation patterns involved in blast induced astrogliosis and cognitive impairments. Further understanding of their role in the injury progression may lead to novel therapeutic targets. PMID:27551260

  17. Selective cleavage enhanced by acetylating the side chain of lysine.

    Science.gov (United States)

    Fu, Leixiaomeng; Chen, Tingting; Xue, Gaiqing; Zu, Lily; Fang, Weihai

    2013-01-01

    Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues. PMID:23303756

  18. PURIFICATION OF L-METHIONINE AND N-ACETYL-D-METHIONINE FROM THE MIXTURE OF ENZYMATICALLY DEACYLATED N-ACETYL-DL-METHIONINE

    Institute of Scientific and Technical Information of China (English)

    YAN Xiaomin; ZHAO Lin; SHAO Jianhui; TAN Xin; SONG Zhengxiao

    2004-01-01

    N-acetyl-D-methionine, NaAc and the remains of N-acetyl-L-methionine dramatically affect the purification of L-methionine when purified from the mixture of enzymatically deacylated N-acetyl-DL-methionine, leading to a low yield conventionally. Here, this paper reports a successful separation and purification of both L-methionine and N-acetyl-D-methionine by an H ion-exchange column. The pH, L-Met concentration and the ratio between the content of sodium cation and the ion-exchange capacity were optimized to obtain the maximum yield. Experimental results indicate that, under the optimized conditions, the yields of L-methionine and N-acetyl-D-methionine can reach as high as 85% and 75%, respectively.

  19. Cell differentiation along multiple pathways accompanied by changes in histone acetylation status.

    Science.gov (United States)

    Legartová, Soňa; Kozubek, Stanislav; Franek, Michal; Zdráhal, Zbyněk; Lochmanová, Gabriela; Martinet, Nadine; Bártová, Eva

    2014-04-01

    Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.

  20. Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus.

    Science.gov (United States)

    Feng, Y; Rustan, T D; Ferguson, R J; Doll, M A; Hein, D W

    1994-01-01

    Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures. PMID:8291051

  1. Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH Screen

    Directory of Open Access Journals (Sweden)

    Ednalise Santiago

    2016-05-01

    Full Text Available Nonmuscle myosin type II (Myo1p is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2 or mass spectrometry (AP-MS (Abp1. The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.

  2. Mice lacking myosin IXb, an inflammatory bowel disease susceptibility gene, have impaired intestinal barrier function and superficial ulceration in the ileum.

    Science.gov (United States)

    Hegan, Peter S; Chandhoke, Surjit K; Barone, Christina; Egan, Marie; Bähler, Martin; Mooseker, Mark S

    2016-04-01

    Genetic studies have implicated MYO9B, which encodes myosin IXb (Myo9b), a motor protein with a Rho GTPase activating domain (RhoGAP), as a susceptibility gene for inflammatory bowel disease (IBD). Moreover, we have recently shown that knockdown of Myo9b in an intestinal epithelial cell line impairs wound healing and barrier function. Here, we investigated whether mice lacking Myo9b have impaired intestinal barrier function and features of IBD. Myo9b knock out (KO) mice exhibit impaired weight gain and fecal occult blood (indicator of gastrointestinal bleeding), and increased intestinal epithelial cell apoptosis could be detected along the entire intestinal axis. Histologic analysis revealed intestinal mucosal damage, most consistently observed in the ileum, which included superficial ulceration and neutrophil infiltration. Focal lesions contained neutrophils and ultrastructural examination confirmed epithelial discontinuity and the deposition of extracellular matrix. We also observed impaired mucosal barrier function in KO mice. Transepithelial electrical resistance of KO ileum is >3 fold less than WT ileum. The intestinal mucosa is also permeable to high molecular weight dextran, presumably due to the presence of mucosal surface ulcerations. There is loss of tight junction-associated ZO-1, decreased lateral membrane associated E-cadherin, and loss of terminal web associated cytokeratin filaments. Consistent with increased Rho activity in the KO, there is increased subapical expression of activated myosin II (Myo2) based on localization of phosphorylated Myo2 regulatory light chain. Except for a delay in disease onset in the KO, no difference in dextran sulfate sodium-induced colitis and lethality was observed between wild-type and Myo9b KO mice.

  3. A random sequential mechanism of aminoglycoside acetylation by Mycobacterium tuberculosis Eis protein.

    Directory of Open Access Journals (Sweden)

    Oleg V Tsodikov

    Full Text Available An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis protein from Mycobacterium tuberculosis (Mt is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed.

  4. 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

  5. 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.

  6. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.

    Science.gov (United States)

    Knight, Peter J; Thirumurugan, Kavitha; Xu, Yuhui; Wang, Fei; Kalverda, Arnout P; Stafford, Walter F; Sellers, James R; Peckham, Michelle

    2005-10-14

    Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke. PMID:16030012

  7. Acetylated starch nanocrystals: Preparation and antitumor drug delivery study.

    Science.gov (United States)

    Xiao, Huaxi; Yang, Tao; Lin, Qinlu; Liu, Gao-Qiang; Zhang, Lin; Yu, Fengxiang; Chen, Yuejiao

    2016-08-01

    In this study, we developed a new nanoparticulate system for acetylated starch nanocrystals (ASN) using broken rice. ASN with different degrees of substitution (DS) of 0.04, 0.08 and 0.14 were prepared using acetic anhydride as acetylating agent through reaction with starch nanocrystals (SN). The resulting ASN were investigated for the capability to load and release doxorubicin hydrochloride (DOX), and the antitumor activities of DOX-loaded SN and DOX-loaded ASN were evaluated as potential drug delivery systems for cancer therapy. Cellular uptake and cytotoxicity of nanocrystals and the DOX-loaded nanocrystals were investigated using fluorescence microscopy and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. Compared with acetylated starches (AS) and native starches (NS), ASN with DS 0.14 loaded up to 6.07% of DOX with a higher loading efficiency of 91.1% and had steadier drug-release rates. Toxicity analysis using the rat hepatocytes model suggested that ASN was biocompatible and could be used for drug delivery. Furthermore, ASN were taken up by cancer cells in vitro and significantly enhanced the cytotoxicity of DOX against HeLa human cervical carcinoma cells. The IC50 value of DOX-loaded ASN-DS 0.14 was 3.8μg/mL for 24h of treatment, which was significantly lower than that of free DOX (21μg/mL). These results indicate that the prepared ASN using broken rice is a promising vehicle for the controlled delivery of DOX for cancer therapy. PMID:27156696

  8. Acetyl hexapeptide-3 in a cosmetic formulation acts on skin mechanical properties - clinical study

    OpenAIRE

    Kassandra Azevedo Tadini; Daiane Garcia Mercurio; Patrícia Maria Berardo Gonçalves Maia Campos

    2015-01-01

    abstract Acetyl hexapeptide-3 has been used in anti-aging topical formulations aimed at improving skin appearance. However, few basic studies address its effects on epidermis and dermis, when vehiculated in topical formulations. Thus, the objective of this study was to determine the clinical efficacy of acetyl hexapeptide-3 using biophysical techniques. For this purpose, formulations with and without acetyl hexapeptide-3 were applied to the ventral forearm and the face area of forty female vo...

  9. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    Energy Technology Data Exchange (ETDEWEB)

    Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

  10. p53 targets simian virus 40 large T antigen for acetylation by CBP.

    Science.gov (United States)

    Poulin, Danielle L; Kung, Andrew L; DeCaprio, James A

    2004-08-01

    Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action with p53. We demonstrate that SV40 T Ag is acetylated in vivo in a p53-dependent manner and T Ag acetylation is largely mediated by CBP. The acetylation of T Ag is dependent on its interaction with p53 and on p53's interaction with CBP. We have mapped the site of acetylation on T Ag to the C-terminal lysine residue 697. This acetylation site is conserved between the T antigens of the human polyomaviruses JC and BK, which are also known to interact with p53. We show that both JC and BK T antigens are also acetylated at corresponding sites in vivo. While other proteins are known to be acetylated by CBP/p300, none are known to depend on p53 for acetylation. T Ag acetylation may provide a regulatory mechanism for T Ag binding to a cellular factor or play a role in another aspect of T Ag function. PMID:15254196

  11. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Juhyung; Yun, Nuri; Kim, Chiho [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Song, Min-Young; Park, Kang-Sik [Department of Physiology and Biomedical Science Institute, Kyung Hee University School of Medicine, Seoul 130-701 (Korea, Republic of); Oh, Young J., E-mail: yjoh@yonsei.ac.kr [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of)

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  12. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells

    International Nuclear Information System (INIS)

    Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium. (author)

  13. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    Science.gov (United States)

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  14. Synthesis of Andrographolide Glucopyranoside and Selective Cleavage of O-acetyl Groups in Sugar Moiety

    Institute of Scientific and Technical Information of China (English)

    WANG Shao-Min; LIU Hong-Min

    2008-01-01

    Andrographolide glucopyranosides were synthesized from andrographolide and tetra-O-acetyl-β-D-glucopyranosyl bromide via a Koenigs-Knorr reaction and deacetylation with a moderate deacetylation reagent dibutyltin oxide in methanol for the first time.The structures of the andrographolide derivatives were confirmed by IR, NMR,and HRMS.Deprotection of the acetylated andrographolide glucopyranoside with dibutyltin oxide in methanol selectively removed all acetyl groups of the sugar moiety, whereas the acetyl group of the andrographolide part and the base- or acid-sensitive functional groups were retained.

  15. Meat consumption, N-acetyl transferase 1 and 2 polymorphism and risk of breast cancer, in Danish postmenopausal women

    DEFF Research Database (Denmark)

    Egeberg, Rikke; Olsen, Anja; Autrup, Herman;

    2008-01-01

    total meat intake and red meat intake and breast cancer risk were confined to intermediate/fast N-acetyl transferase 2 acetylators (P-interaction=0.03 and 0.04). Our findings support an association between meat consumption and breast cancer risk and that N-acetyl transferase 2 polymorphism has......The aim of this study was to investigate whether polymorphisms in N-acetyl transferase 1 and 2 modify the association between meat consumption and risk of breast cancer. A nested case-control study was conducted among 24697 postmenopausal women included in the 'Diet, Cancer and Health' cohort study...... increment in intake. Compared with slow acetylators, the IRR (95% confidence interval) among fast N-acetyl transferase 1 acetylators was 1.43 (1.03-1.99) and 1.13 (0.83-1.54) among intermediate/fast N-acetyl transferase 2 acetylators. Interaction analyses revealed that the positive associations between...

  16. 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.

  17. 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.

  18. A reactivity-selectivity study of the Friedel-Crafts acetylation of 3,3′-dimethylbiphenyl and the oxidation of the acetyl derivatives

    DEFF Research Database (Denmark)

    Titinchi, Salam JJ; Kamounah, Fadhil S.; Abbo, Hanna S.;

    2012-01-01

    Background: Friedel-Crafts acetylation is an important route to aromatic ketones, in research laboratories and in industry. The acetyl derivatives of 3,3'-dimethylbiphenyl (3,3'-dmbp) have applications in the field of liquid crystals and polymers and may be oxidized to the dicarboxylic acids...... converted to the carboxylic acids by hypochlorite oxidation. The relative stabilities of the isomeric products and the corresponding s-complexes were studied by DFT calculations and the data indicated that mono-and diacetylation followed different mechanisms. Conclusions: Friedel-Crafts acetylation of 3...

  19. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway

    OpenAIRE

    Betapudi, Venkaiah; Lominadze, George; Hsi, Linda; Willard, Belinda; Wu, Meifang; McCrae, Keith R

    2013-01-01

    Activation of endothelial cells by anti-β2GPI antibodies causes myosin RLC phosphorylation, leading to actin-myosin association.In response to anti-β2GPI antibodies, release of endothelial microparticles, but not E-selectin expression, requires actomyosin assembly.

  20. 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...

  1. Distribution of myosin heavy chain isoforms in muscular dystrophy: insights into disease pathology

    Science.gov (United States)

    Beedle, Aaron M

    2016-01-01

    Myosin heavy chain isoforms are an important component defining fiber type specific properties in skeletal muscle, such as oxidative versus glycolytic metabolism, rate of contraction, and fatigability. While the molecular mechanisms that underlie specification of the different fiber types are becoming clearer, how this programming becomes disrupted in muscular dystrophy and the functional consequences of fiber type changes in disease are not fully resolved. Fiber type changes in disease, with specific focus on muscular dystrophies caused by defects in the dystrophin glycoprotein complex, are discussed. PMID:27430020

  2. 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.

  3. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Directory of Open Access Journals (Sweden)

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  4. The dynamic organization of fungal acetyl-CoA carboxylase

    Science.gov (United States)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  5. Kinetics of oxygen-18 exchange between inorganic phosphate and water catalyzed by myosin subfragment 1, using the 18O shift in 31P NMR.

    Science.gov (United States)

    Webb, M R; McDonald, G G; Trentham, D R

    1978-05-10

    The time course of oxygen-18 exchange between [18O]Pi and normal water, catalyzed by myosin subfragment 1 in the presence of MgADP, was followed using the shift in 31P NMR caused by the presence of oxygen-18 bound to the phosphorus. Essentially all molecules of [18O]Pi that bind to the enzyme undergo complete exchange and are released as [16O4]Pi. Exchange probably occurs by formation of myosin.ATP from a myosin.ADP.Pi complex and is rapid relative to release of Pi from this complex. The kinetics of exchange give a value for the rate constant for binding Pi to myosin.ADP of 0.23 M-1 S-1 (pH 8.0, 22 degrees C). This value is consistent with exchange occurring by reversal of the ATP-ase reaction back to the myosin.ATP complex. PMID:641045

  6. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    Science.gov (United States)

    Tsuchiya, Yugo; Pham, Uyen; Hu, Wanzhou; Ohnuma, Shin-Ichi; Gout, Ivan

    2014-01-01

    Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis. PMID:24831956

  7. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Yugo Tsuchiya

    Full Text Available Coenzyme A (CoA is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis.

  8. Strain-Dependent Kinetics of the Myosin Working Stroke, and How They Could Be Probed with Optical-Trap Experiments

    OpenAIRE

    Smith, David; Sleep, John

    2006-01-01

    The strain-dependent kinetics of the myosin working stroke under load is derived from a flat-energy-landscape model for its untethered lever-arm, and compared with other scenarios in the literature. The “flat landscape” scenario is compatible with muscle-fiber experiments, but is more critically relevant to single-myosin experiments with an optically trapped actin filament. In such experiments, the strain dependence of stroke kinetics may be explored by comparing event-averaged and time-avera...

  9. Topography induces differential sensitivity on cancer cell proliferation via Rho-ROCK-Myosin contractility

    Science.gov (United States)

    Chaudhuri, Parthiv Kant; Pan, Catherine Qiurong; Low, Boon Chuan; Lim, Chwee Teck

    2016-01-01

    Although the role of stiffness on proliferative response of cancer cells has been well studied, little is known about the effect of topographic cues in guiding cancer cell proliferation. Here, we examined the effect of topographic cues on cancer cell proliferation using micron scale topographic features and observed that anisotropic features like microgratings at specific dimension could reduce proliferation of non-cancer breast epithelial cells (MCF-10A) but not that for malignant breast cancer cells (MDA-MB-231 and MCF-7). However, isotropic features such as micropillars did not affect proliferation of MCF-10A, indicating that the anisotropic environmental cues are essential for this process. Interestingly, acto-myosin contraction inhibitory drugs, Y-27632 and blebbistatin prevented micrograting-mediated inhibition on proliferation. Here, we propose the concept of Mechanically-Induced Dormancy (MID) where topographic cues could activate Rho-ROCK-Myosin signaling to suppress non-cancerous cells proliferation whereas malignant cells are resistant to this inhibitory barrier and therefore continue uncontrolled proliferation. PMID:26795068

  10. A single-headed fission yeast myosin V transports actin in a tropomyosin-dependent manner.

    Science.gov (United States)

    Tang, Qing; Billington, Neil; Krementsova, Elena B; Bookwalter, Carol S; Lord, Matthew; Trybus, Kathleen M

    2016-07-18

    Myo51, a class V myosin in fission yeast, localizes to and assists in the assembly of the contractile ring, a conserved eukaryotic actomyosin structure that facilitates cytokinesis. Rng8 and Rng9 are binding partners that dictate the cellular localization and function of Myo51. Myo51 was expressed in insect cells in the presence or absence of Rng8/9. Surprisingly, electron microscopy of negatively stained images and hydrodynamic measurements showed that Myo51 is single headed, unlike most class V myosins. When Myo51-Rng8/9 was bound to actin-tropomyosin, two attachment sites were observed: the typical ATP-dependent motor domain attachment and a novel ATP-independent binding of the tail mediated by Rng8/9. A modified motility assay showed that this additional binding site anchors Myo51-Rng8/9 so that it can cross-link and slide actin-tropomyosin filaments relative to one another, functions that may explain the role of this motor in contractile ring assembly. PMID:27432898

  11. 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.

  12. 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.

  13. Temperature-dependent developmental variation in lobster muscle myosin heavy chain isoforms.

    Science.gov (United States)

    Magnay, J L; Holmes, J M; Neil, D M; El Haj, A J

    2003-10-16

    The temperature- and developmental-regulation of myosin heavy chain (MyHC) expression and primary sequence was investigated in the abdominal musculature of developing Homarus gammarus larvae acclimated to 10, 14 and 19+/-1 degrees C. MyHC loop 1 (ATP binding) and loop 2 (actin binding) regions were sequenced and compared. The deduced amino acid sequence of MyHC loop 1 showed a development-related increase in net charge from +1 to +2 between larval stages 1 and 2, which was not temperature-dependent. In post-settled stage 9 larvae, minor shifts in amino acid sequence occurred at 19 degrees C, and corresponded to a significant up-regulation of fast myosin mRNA expression. However, no temperature-specific loop 1 isoforms were detected. The deduced amino acid sequence of MyHC loop 2 was not affected by temperature, and the net charge remained +4 throughout development. These findings contrast to previous studies using the common carp, in which temperature-specific MyHC isoform genes were expressed in response to disparate thermal regimes. This raises the question as to whether arthropods do not express specific temperature isoforms but instead rely on shifts in fibre type to accommodate alterations in thermal environment. PMID:14563558

  14. Lentivirus-Mediated Knockdown of Myosin VI Inhibits Cell Proliferation of Breast Cancer Cell.

    Science.gov (United States)

    Wang, Hong; Wang, Biyun; Zhu, Wei; Yang, Ziang

    2015-10-01

    Myosin VI (MYO6) is a unique member of the myosin superfamily, and almost no experimental studies link MYO6 to tumorigenesis of breast cancer. However, previous microarray data demonstrated that MYO6 was frequently overexpressed in breast cancer tissues. In this study, to further develop its role in breast cancer, endogenous expression of MYO6 was significantly inhibited in breast cancer ZR-75-30 and MDA-MB-231 cells using lentivirus-mediated RNA interference. Quantitative polymerase chain reaction and western blot were applied to detect the expression level of MYO6. Cell viability of both cell lines was measured by methylthiazol tetrazolium and colony formation assays. Besides, cell cycle assay was utilized to acquire the distribution information of cell phase. The results demonstrated that knockdown of MYO6 markedly reduced cell viability and colony formation, as well as suppressed cell cycle progression in breast cancer cells. The results suggested that MYO6 played a vital role in breast cancer cells and might provide useful information for diagnosis and therapy of human breast cancer in future. PMID:26407123

  15. 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.

  16. 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.

  17. Location of the O-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan.

    Science.gov (United States)

    York, W S; Oates, J E; van Halbeek, H; Darvill, A G; Albersheim, P; Tiller, P R; Dell, A

    1988-02-15

    The locations of the O-acetyl substituents on the major nonasaccharide repeating unit of the xyloglucan isolated from sycamore extracellular polysaccharides were determined by a combination of analytical methods, including f.a.b.-m.s. and 1H-n.m.r. spectroscopy. The O-2-linked-beta-D-galactosyl residue of the nonasaccharide was found to be the dominant site of O-acetyl substitution. Both mono-O-acetylated and di-O-acetylated beta-D-galactosyl residues were detected. The degree of O-acetylation of the beta-D-galactosyl residue, was estimated by 1H-n.m.r. spectroscopy to be 55-60% at O-6, 15-20% at O-4, and 20-25% at O-3. 1H-n.m.r. spectroscopy also indicated that approximately 50% of the beta-D-galactosyl residues are mono-O-acetylated, 25-30% are di-O-acetylated, and 20% are not acetylated.

  18. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian;

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 ly...

  19. A dysregulated acetyl/SUMO switch of FXR promotes hepatic inflammation in obesity.

    Science.gov (United States)

    Kim, Dong-Hyun; Xiao, Zhen; Kwon, Sanghoon; Sun, Xiaoxiao; Ryerson, Daniel; Tkac, David; Ma, Ping; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Zhou, Edward; Xu, H Eric; Palvimo, Jorma J; Chen, Lin-Feng; Kemper, Byron; Kemper, Jongsook Kim

    2015-01-13

    Acetylation of transcriptional regulators is normally dynamically regulated by nutrient status but is often persistently elevated in nutrient-excessive obesity conditions. We investigated the functional consequences of such aberrantly elevated acetylation of the nuclear receptor FXR as a model. Proteomic studies identified K217 as the FXR acetylation site in diet-induced obese mice. In vivo studies utilizing acetylation-mimic and acetylation-defective K217 mutants and gene expression profiling revealed that FXR acetylation increased proinflammatory gene expression, macrophage infiltration, and liver cytokine and triglyceride levels, impaired insulin signaling, and increased glucose intolerance. Mechanistically, acetylation of FXR blocked its interaction with the SUMO ligase PIASy and inhibited SUMO2 modification at K277, resulting in activation of inflammatory genes. SUMOylation of agonist-activated FXR increased its interaction with NF-κB but blocked that with RXRα, so that SUMO2-modified FXR was selectively recruited to and trans-repressed inflammatory genes without affecting FXR/RXRα target genes. A dysregulated acetyl/SUMO switch of FXR in obesity may serve as a general mechanism for diminished anti-inflammatory response of other transcriptional regulators and provide potential therapeutic and diagnostic targets for obesity-related metabolic disorders. PMID:25425577

  20. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.