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Sample records for actin-bound myosin heads

  1. Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

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    Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

    2010-01-01

    SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

  2. The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

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    Banerjee, Chaity; Hu, Zhongjun; Huang, Zhong; Warrington, J Anthony; Taylor, Dianne W; Trybus, Kathleen M; Lowey, Susan; Taylor, Kenneth A

    2017-12-01

    Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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    Katsuyuki Shiroguchi

    2011-04-01

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

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

    International Nuclear Information System (INIS)

    Minoda, Hiroki; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-01-01

    Research highlights: → We succeeded in recording structural changes of hydrated myosin cross-bridges. → We succeeded in position-marking the cross-bridges with site-directed antibodies. → We recorded cross-bridge movement at different regions in individual cross-bridge. → The movement was smallest at the cross-bridge-subfragment two boundary. → 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.

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

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    Mirco Müller

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

  6. Biased Brownian motion mechanism for processivity and directionality of single-headed myosin-VI.

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    Iwaki, Mitsuhiro; Iwane, Atsuko Hikikoshi; Ikebe, Mitsuo; Yanagida, Toshio

    2008-01-01

    Conventional form to function as a vesicle transporter is not a 'single molecule' but a coordinated 'two molecules'. The coordinated two molecules make it complicated to reveal its mechanism. To overcome the difficulty, we adopted a single-headed myosin-VI as a model protein. Myosin-VI is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. However, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting doubt on its processivity. Using single molecule techniques, we show that green fluorescent protein (GFP)-fused single-headed myosin-VI does not move processively. However, when coupled to a 200 nm polystyrene bead (comparable to an intracellular vesicle in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40 nm) steps. Furthermore, we found that a single-headed myosin-VI-bead complex moved more processively in a high-viscous solution (40-fold higher than water) similar to cellular environment. Because diffusion of the bead is 60-fold slower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing the head's rebinding following detachment and supporting processive movement of the bead-monomer complex. This investigation will help us understand how molecular motors utilize Brownian motion in cells.

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

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    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.

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

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    Kumakura, Michiko; Kawaguchi, Atsushi; Nagata, Kyosuke

    2015-01-01

    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

  9. Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications

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    Haruo Sugi

    2018-05-01

    Full Text Available The most straightforward way to get information on the performance of individual myosin heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC. The EC enables us to visualize and record ATP-induced myosin head movement in hydrated skeletal muscle myosin filaments. When actin filaments are absent, myosin heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, myosin heads are found to move away from, but not towards, the bare region, indicating that myosin heads perform a recovery stroke (average amplitude, 6 nm. After exhaustion of ATP, myosin heads return to their neutral position. In the actin–myosin filament mixture, myosin heads form rigor actin myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the myosin head catalytic domain (CAD, respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, myosin heads return to their neutral position after exhaustion of ATP.

  10. Actin and myosin contribute to mammalian mitochondrial DNA maintenance

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    Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

    2011-01-01

    Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. PMID:21398640

  11. Stress generation by myosin minifilaments in actin bundles

    International Nuclear Information System (INIS)

    Dasanayake, Nilushi L; Carlsson, Anders E

    2013-01-01

    Forces and stresses generated by the action of myosin minifilaments are analyzed in idealized computer-generated actin bundles, and compared to results for isotropic actin networks. The bundles are generated as random collections of actin filaments in two dimensions with constrained orientations, crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending and stretching of actin filaments, which accomodate minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself. (paper)

  12. Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.

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    Hariadi, Rizal F; Appukutty, Abhinav J; Sivaramakrishnan, Sivaraj

    2016-09-27

    Nature has evolved molecular motors that are critical in cellular processes occurring over broad time scales, ranging from seconds to years. Despite the importance of the long-term behavior of molecular machines, topics such as enzymatic lifetime are underexplored due to the lack of a suitable approach for monitoring motor activity over long time periods. Here, we developed an "O"-shaped Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale DNA nanotube rings with precise arrangements of myosin VI to trap gliding actin filaments. This circular gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 motors per ring) multiple times. First, we systematically characterized the formation of DNA nanotubes rings with 4, 6, 8, and 10 helix circumferences. Individual actin filaments glide along the nanotube rings with high processivity for up to 12.8 revolutions or 11 min in run time. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 μm). As a model application of OMEGA, we then analyze motor-based mechanical influence on "stop-and-go" gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes.

  13. Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers.

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    Arata, T

    1990-07-20

    Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.

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

    International Nuclear Information System (INIS)

    Sellers, James R.; Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-01-01

    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

  15. Unconstrained steps of myosin VI appear longest among known molecular motors.

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    Ali, M Yusuf; Homma, Kazuaki; Iwane, Atsuko Hikikoshi; Adachi, Kengo; Itoh, Hiroyasu; Kinosita, Kazuhiko; Yanagida, Toshio; Ikebe, Mitsuo

    2004-06-01

    Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V.

  16. Localization of Myosin and Actin in the Pelage and Whisker Hair Follicles of Rat

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    Morioka, Kiyokazu; Matsuzaki, Toshiyuki; Takata, Kuniaki

    2006-01-01

    The combined effects of myosin II and actin enable muscle and nonmuscle cells to generate forces required for muscle contraction, cell division, cell migration, cellular morphological changes, the maintenance of cellular tension and polarity, and so on. However, except for the case of muscle contraction, the details are poorly understood. We focus on nonmuscle myosin and actin in the formation and maintenance of hair and skin, which include highly active processes in mammalian life with respect to the cellular proliferation, differentiation, and movement. The localization of nonmuscle myosin II and actin in neonatal rat dorsal skin, mystacial pad, hair follicles, and vibrissal follicles was studied by immunohistochemical technique to provide the basis for the elucidation of the roles of these proteins. Specificities of the antibodies were verified by using samples from the relevant tissues and subjecting them to immunoblotting test prior to morphological analyses. The myosin and actin were abundant and colocalized in the spinous and granular layers but scarce in the basal layer of the dorsal and mystacial epidermis. In hair and vibrissal follicles, nonmuscle myosin and actin were colocalized in the outer root sheath and some hair matrix cells adjoining dermal papillae. In contrast, most areas of the inner root sheath and hair matrix appeared to comprise very small amounts of myosin and actin. Hair shaft may comprise significant myosin during the course of its keratinization. These results suggest that the actin-myosin system plays a part in cell movement, differentiation, protection and other key functions of skin and hair cells

  17. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

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    Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

    2017-06-30

    Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s -1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s -1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin.

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    Kohei Iwase

    Full Text Available We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

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

    International Nuclear Information System (INIS)

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2010-01-01

    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 o /12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127 o range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from

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

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    Shenping Wu

    2010-09-01

    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

  1. Conformational distributions and proximity relationships in the rigor complex of actin and myosin subfragment-1.

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    Nyitrai, M; Hild, G; Lukács, A; Bódis, E; Somogyi, B

    2000-01-28

    Cyclic conformational changes in the myosin head are considered essential for muscle contraction. We hereby show that the extension of the fluorescence resonance energy transfer method described originally by Taylor et al. (Taylor, D. L., Reidler, J., Spudich, J. A., and Stryer, L. (1981) J. Cell Biol. 89, 362-367) allows determination of the position of a labeled point outside the actin filament in supramolecular complexes and also characterization of the conformational heterogeneity of an actin-binding protein while considering donor-acceptor distance distributions. Using this method we analyzed proximity relationships between two labeled points of S1 and the actin filament in the acto-S1 rigor complex. The donor (N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate) was attached to either the catalytic domain (Cys-707) or the essential light chain (Cys-177) of S1, whereas the acceptor (5-(iodoacetamido)fluorescein) was attached to the actin filament (Cys-374). In contrast to the narrow positional distribution (assumed as being Gaussian) of Cys-707 (5 +/- 3 A), the positional distribution of Cys-177 was found to be broad (102 +/- 4 A). Such a broad positional distribution of the label on the essential light chain of S1 may be important in accommodating the helically arranged acto-myosin binding relative to the filament axis.

  2. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  3. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    OpenAIRE

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed ...

  4. Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

    Energy Technology Data Exchange (ETDEWEB)

    Mijailovich, Srboljub M.; Kayser-Herold, Oliver; Stojanovic, Boban; Nedic, Djordje; Irving, Thomas C.; Geeves, MA (Harvard); (IIT); (U. Kent); (Kragujevac)

    2016-11-18

    The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to

  5. CHARACTERIZATION OF TIGHTLY-ASSOCIATED SMOOTH MUSCLE MYOSIN-MYOSIN LIGHT CHAIN KINASE-CALMODULIN COMPLEXES*

    OpenAIRE

    Hong, Feng; Haldeman, Brian D.; John, Olivia A.; Brewer, Paul D.; Wu, Yi-Ying; Ni, Shaowei; Wilson, David P.; Walsh, Michael P.; Baker, Jonathan E.; Cremo, Christine R.

    2009-01-01

    A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen t...

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

    Science.gov (United States)

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

    2010-08-06

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

  7. Probing muscle myosin motor action: x-ray (m3 and m6) interference measurements report motor domain not lever arm movement.

    Science.gov (United States)

    Knupp, Carlo; Offer, Gerald; Ranatunga, K W; Squire, John M

    2009-07-10

    The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.

  8. Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture.

    Science.gov (United States)

    Kagawa, Maiko; Sato, Naruki; Obinata, Takashi

    2006-11-01

    Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.

  9. 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.

  10. Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres.

    Science.gov (United States)

    Avrova, Stanislava V; Rysev, Nikita A; Matusovsky, Oleg S; Shelud'ko, Nikolay S; Borovikov, Yurii S

    2012-05-01

    The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

    Science.gov (United States)

    Kuroda, Taruho S; Fukuda, Mitsunori

    2005-01-01

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  12. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    International Nuclear Information System (INIS)

    Vu, N.D.; Wagner, P.D.

    1987-01-01

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32 P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca 2+ - and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation

  13. Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: Mediation by cyclic AMP

    International Nuclear Information System (INIS)

    Egan, J.J.; Gronowicz, G.; Rodan, G.A.

    1991-01-01

    Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP

  14. Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

    Directory of Open Access Journals (Sweden)

    Kanji Oshima

    Full Text Available The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation

  15. The Effect of Experimental Hyperthyroidism on Characteristics of Actin-Myosin Interaction in Fast and Slow Skeletal Muscles.

    Science.gov (United States)

    Kopylova, G V; Shchepkin, D V; Bershitsky, S Y

    2018-05-01

    The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin-myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.

  16. Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle

    Science.gov (United States)

    Tanner, Bertrand C.W.; McNabb, Mark; Palmer, Bradley M.; Toth, Michael J.; Miller, Mark S.

    2014-01-01

    Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either i) uniformly, from the Z-line end of thick-filaments, or ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulation also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere. PMID:24486373

  17. The fungal myosin I is essential for Fusarium toxisome formation.

    Directory of Open Access Journals (Sweden)

    Guangfei Tang

    2018-01-01

    Full Text Available Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1, the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

  18. The fungal myosin I is essential for Fusarium toxisome formation.

    Science.gov (United States)

    Tang, Guangfei; Chen, Yun; Xu, Jin-Rong; Kistler, H Corby; Ma, Zhonghua

    2018-01-01

    Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

  19. Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

    Science.gov (United States)

    Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

    2008-02-01

    To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

  20. Flexural Stiffness of Myosin Va Subdomains as Measured from Tethered Particle Motion

    Science.gov (United States)

    Michalek, Arthur J.; Kennedy, Guy G.; Warshaw, David M.; Ali, M. Yusuf

    2015-01-01

    Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor's structure. Specifically, changes in calcium may regulate motor processivity by altering the motor's lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor's flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network. PMID:26770194

  1. Coupling between myosin head conformation and the thick filament backbone structure.

    Science.gov (United States)

    Hu, Zhongjun; Taylor, Dianne W; Edwards, Robert J; Taylor, Kenneth A

    2017-12-01

    The recent high-resolution structure of the thick filament from Lethocerus asynchronous flight muscle shows aspects of thick filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the thick filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the thick filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin filament followed by the free head when force production occurs. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, S F; Hoffmann, E K

    2002-01-01

    effects on F-actin. The subsequent F-actin depolymerization, however, appeared MLCK- and PKC-dependent, and the initial swelling-induced F-actin depolymerization was MLCK-dependent; both effects were apparently secondary to kinase-mediated effects on cell volume changes. NHE1 in EATC is activated both....... Moreover, Rho kinase inhibition did not significantly affect NHE1 activation, neither by shrinkage nor by CL-A. Implications for the possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, and cell volume regulation are discussed....

  3. Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

    Directory of Open Access Journals (Sweden)

    Thomas P Burghardt

    Full Text Available Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function. A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N

  4. N-terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size

    Science.gov (United States)

    Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Burghardt, Thomas P.

    2016-01-01

    Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ~19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method. PMID:26671638

  5. Why muscle is an efficient shock absorber.

    Directory of Open Access Journals (Sweden)

    Michael A Ferenczi

    Full Text Available Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.

  6. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen

  7. A Novel Alpha Cardiac Actin (ACTC1 Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects.

    Directory of Open Access Journals (Sweden)

    Céline Augière

    Full Text Available A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects, conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5.A set of 399 poly(AC markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1 among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys and p.(Met125Val which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser,p.(Asp313His and p.(Arg314His which result in diverse cardiomyopathies and are located in a totally different interaction surface.Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin.

  8. Myosin light chain phosphorylation is critical for adaptation to cardiac stress.

    Science.gov (United States)

    Warren, Sonisha A; Briggs, Laura E; Zeng, Huadong; Chuang, Joyce; Chang, Eileen I; Terada, Ryota; Li, Moyi; Swanson, Maurice S; Lecker, Stewart H; Willis, Monte S; Spinale, Francis G; Maupin-Furlowe, Julie; McMullen, Julie R; Moss, Richard L; Kasahara, Hideko

    2012-11-27

    Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

  9. In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics.

    Science.gov (United States)

    Mishra, Mithilesh; Kashiwazaki, Jun; Takagi, Tomoko; Srinivasan, Ramanujam; Huang, Yinyi; Balasubramanian, Mohan K; Mabuchi, Issei

    2013-07-01

    Cytokinesis in many eukaryotes involves the contraction of an actomyosin-based contractile ring. However, the detailed mechanism of contractile ring contraction is not fully understood. Here, we establish an experimental system to study contraction of the ring to completion in vitro. We show that the contractile ring of permeabilized fission yeast cells undergoes rapid contraction in an ATP- and myosin-II-dependent manner in the absence of other cytoplasmic constituents. Surprisingly, neither actin polymerization nor its disassembly is required for contraction of the contractile ring, although addition of exogenous actin-crosslinking proteins blocks ring contraction. Using contractile rings generated from fission yeast cytokinesis mutants, we show that not all proteins required for assembly of the ring are required for its contraction in vitro. Our work provides the beginnings of the definition of a minimal contraction-competent cytokinetic ring apparatus.

  10. Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain.

    Science.gov (United States)

    Bódis, Emöke; Strambini, Giovanni B; Gonnelli, Margherita; Málnási-Csizmadia, András; Somogyi, Béla

    2004-08-01

    The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.

  11. Myosin VIIa as a common component of cilia and microvilli.

    Science.gov (United States)

    Wolfrum, U; Liu, X; Schmitt, A; Udovichenko, I P; Williams, D S

    1998-01-01

    The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

  12. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-01-01

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin

  13. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

    Science.gov (United States)

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells

    Science.gov (United States)

    Lehmann, Maik J.; Sherer, Nathan M.; Marks, Carolyn B.; Pypaert, Marc; Mothes, Walther

    2005-01-01

    Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, “surfing” toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body. PMID:16027225

  15. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells.

    Science.gov (United States)

    Lehmann, Maik J; Sherer, Nathan M; Marks, Carolyn B; Pypaert, Marc; Mothes, Walther

    2005-07-18

    Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

  16. Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

    Directory of Open Access Journals (Sweden)

    Orion D Weiner

    2006-02-01

    Full Text Available Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes, scaffolded by hematopoietic protein 1 (Hem-1, that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b substantially weakens Rac activation and phosphatidylinositol-(3,4,5-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5-tris-phosphate/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

  17. Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

    Science.gov (United States)

    Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

    2018-05-08

    The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

  18. Botulinum Toxin Type A Inhibits α-Smooth Muscle Actin and Myosin II Expression in Fibroblasts Derived From Scar Contracture.

    Science.gov (United States)

    Chen, Minliang; Yan, Tongtong; Ma, Kui; Lai, Linying; Liu, Chang; Liang, Liming; Fu, Xiaobing

    2016-09-01

    Scar contracture (SC) is one of the most common complications resulting from major burn injuries. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Recent reports suggest that botulinum toxin type A (BTXA) is effective at reducing SC clinically, but the molecular mechanism for this action is unknown. α-Smooth muscle actin (α-SMA) and myosin II are the main components of stress fibers, which are the contractile structures of fibroblasts. The effects of BTXA on α-SMA and myosin II in SC are still unknown. This study aimed to explore the effect of BTXA on α-SMA and myosin II expression in fibroblasts derived from SC and to elucidate its actual mechanism further. Fibroblasts were isolated from tissue specimens of SC. Fibroblasts were cultured in Dulbecco modified Eagle medium with different concentrations of BTXA and their proliferation was analyzed through the tetrazolium-based colorimetric method at 1, 4, and 7 days. Proteins of α-SMA and myosin II were checked using Western blot in fibroblasts treated with different concentrations of BTXA at 1, 4, and 7 days. Fibroblasts without BTXA treatment had a higher proliferation than that in other groups, which indicated that the proliferation of fibroblasts was significantly inhibited by BTXA (P < 0.05). Proteins of α-SMA and myosin II between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (P < 0.05). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from SC and reduced the expression of α-SMA and myosin II, which provided theoretical support for the application of BTXA to control SC.

  19. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

    Science.gov (United States)

    Suggs, Jennifer A; Melkani, Girish C; Glasheen, Bernadette M; Detor, Mia M; Melkani, Anju; Marsan, Nathan P; Swank, Douglas M; Bernstein, Sanford I

    2017-06-01

    Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro

  20. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects

    Directory of Open Access Journals (Sweden)

    Jennifer A. Suggs

    2017-06-01

    Full Text Available Individuals with inclusion body myopathy type 3 (IBM3 display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in

  1. Model for adhesion clutch explains biphasic relationship between actin flow and traction at the cell leading edge

    Science.gov (United States)

    Craig, Erin M.; Stricker, Jonathan; Gardel, Margaret L.; Mogilner, Alex

    2015-01-01

    Cell motility relies on the continuous reorganization of a dynamic actin-myosin-adhesion network at the leading edge of the cell, in order to generate protrusion at the leading edge and traction between the cell and its external environment. We analyze experimentally measured spatial distributions of actin flow, traction force, myosin density, and adhesion density in control and pharmacologically perturbed epithelial cells in order to develop a mechanical model of the actin-adhesion-myosin self-organization at the leading edge. A model in which the F-actin network is treated as a viscous gel, and adhesion clutch engagement is strengthened by myosin but weakened by actin flow, can explain the measured molecular distributions and correctly predict the spatial distributions of the actin flow and traction stress. We test the model by comparing its predictions with measurements of the actin flow and traction stress in cells with fast and slow actin polymerization rates. The model predicts how the location of the lamellipodium-lamellum boundary depends on the actin viscosity and adhesion strength. The model further predicts that the location of the lamellipodium-lamellum boundary is not very sensitive to the level of myosin contraction. PMID:25969948

  2. Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

    Science.gov (United States)

    Alamo, Lorenzo; Koubassova, Natalia; Pinto, Antonio; Gillilan, Richard; Tsaturyan, Andrey; Padrón, Raúl

    2017-10-01

    The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca 2+ -activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

  3. Myosin-II sets the optimal response time scale of chemotactic amoeba

    Science.gov (United States)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Bodenschatz, Eberhard; Beta, Carsten

    2014-03-01

    The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba. Institute of Physics und Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany.

  4. Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

    International Nuclear Information System (INIS)

    Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

    1987-01-01

    In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

  5. A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress.

    Science.gov (United States)

    Wilkie, Adrian R; Sharma, Mayuri; Pesola, Jean M; Ericsson, Maria; Fernandez, Rosio; Coen, Donald M

    2018-03-15

    Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress. IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for

  6. Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

    Science.gov (United States)

    Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier

    2015-01-01

    The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

  7. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    OpenAIRE

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin sub...

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

    Science.gov (United States)

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

    2016-12-12

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

  9. Myosin X is recruited to nascent focal adhesions at the leading edge and induces multi-cycle filopodial elongation.

    Science.gov (United States)

    He, Kangmin; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Watanabe, Tomonobu M; Ikebe, Mitsuo

    2017-10-20

    Filopodia protrude from the leading edge of cells and play important roles in cell motility. Here we report the mechanism of myosin X (encoded by Myo10)-induced multi-cycle filopodia extension. We found that actin, Arp2/3, vinculin and integrin-β first accumulated at the cell's leading edge. Myosin X was then gathered at these sites, gradually clustered by lateral movement, and subsequently initiated filopodia formation. During filopodia extension, we found the translocation of Arp2/3 and integrin-β along filopodia. Arp2/3 and integrin-β then became localized at the tip of filopodia, from where myosin X initiated the second extension of filopodia with a change in extension direction, thus producing long filopodia. Elimination of integrin-β, Arp2/3 and vinculin by siRNA significantly attenuated the myosin-X-induced long filopodia formation. We propose the following mechanism. Myosin X accumulates at nascent focal adhesions at the cell's leading edge, where myosin X promotes actin convergence to create the base of filopodia. Then myosin X moves to the filopodia tip and attracts integrin-β and Arp2/3 for further actin nucleation. The tip-located myosin X then initiates the second cycle of filopodia elongation to produce the long filopodia.

  10. The Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an In Vitro Model System

    OpenAIRE

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

    2013-01-01

    During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca2+-CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vi...

  11. Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

    Science.gov (United States)

    Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin

    2012-01-01

    Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

  12. Force Exertion and Transmission in Cross-Linked Actin Networks

    Science.gov (United States)

    Stam, Samantha

    Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

  13. Allosteric communication in myosin V: from small conformational changes to large directed movements.

    Directory of Open Access Journals (Sweden)

    M Cecchini

    Full Text Available The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

  14. Strong Dependence of Hydration State of F-Actin on the Bound Mg(2+)/Ca(2+) Ions.

    Science.gov (United States)

    Suzuki, Makoto; Imao, Asato; Mogami, George; Chishima, Ryotaro; Watanabe, Takahiro; Yamaguchi, Takaya; Morimoto, Nobuyuki; Wazawa, Tetsuichi

    2016-07-21

    Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f2 (fw). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal Dhyme (≡ (f1 - fw)δ1/(fwδw)) of F-actin is stronger than that of G-actin, where δw is DR-amplitude of bulk solvent and δ1 is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca(2+)) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg(2+)). Moreover, in the presence of 2 mM Mg(2+), the hydration state of F-actin is changed by adding a small fraction of Ca(2+) (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera.

  15. Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

    International Nuclear Information System (INIS)

    Ajtai, K.; Burghardt, T.P.

    1989-01-01

    The authors describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. They determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [ 14 C]iodoacetamide or enzymatic activity measurements. They characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization. They find that the SH2 probe distinguishes the different states of the fiber such that rigor and MgADP are ordered and maintain a similar orientation throughout the excitation wavelength domain. The relaxed cross-bridge is ordered and has an orientation that is distinct from the orientation of the cross-bridge in rigor and MgADP over the entire wavelength domain. The active isometric cross-bridge is also oriented differently from the other states, suggesting the presence of a predominant actin-bound cross-bridge state that precedes the power stroke during muscle contraction

  16. Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

    International Nuclear Information System (INIS)

    Bhadriraju, Kiran; Hansen, Linda K.

    2004-01-01

    Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation

  17. 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

    2002-01-01

    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. PMID:12485990

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

    Science.gov (United States)

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

    1998-04-21

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

  19. 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.

  20. 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.

  1. IFT88 influences chondrocyte actin organization and biomechanics.

    Science.gov (United States)

    Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

    2016-03-01

    Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Prabuddha Gupta

    Full Text Available Myosin-1 (Myo1 represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1-8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP and Myo2 (by Blebbistatin lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm, is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

  3. Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo.

    Science.gov (United States)

    Morita, Toshiyuki; Tsuchiya, Akiko; Sugimoto, Masazumi

    2011-09-01

    Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

  4. Selective, retrieval-independent disruption of methamphetamine-associated memory by actin depolymerization.

    Science.gov (United States)

    Young, Erica J; Aceti, Massimiliano; Griggs, Erica M; Fuchs, Rita A; Zigmond, Zachary; Rumbaugh, Gavin; Miller, Courtney A

    2014-01-15

    Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder. There is a growing consensus that memory is supported by structural and functional plasticity driven by F-actin polymerization in postsynaptic dendritic spines at excitatory synapses. However, the mechanisms responsible for the long-term maintenance of memories, after consolidation has occurred, are largely unknown. Conditioned place preference (n = 112) and context-induced reinstatement of self-administration (n = 19) were used to assess the role of F-actin polymerization and myosin II, a molecular motor that drives memory-promoting dendritic spine actin polymerization, in the maintenance of METH-associated memories and related structural plasticity. Memories formed through association with METH but not associations with foot shock or food reward were disrupted by a highly-specific actin cycling inhibitor when infused into the amygdala during the postconsolidation maintenance phase. This selective effect of depolymerization on METH-associated memory was immediate, persistent, and did not depend upon retrieval or strength of the association. Inhibition of non-muscle myosin II also resulted in a disruption of METH-associated memory. Thus, drug-associated memories seem to be actively maintained by a unique form of cycling F-actin driven by myosin II. This finding provides a potential therapeutic approach for the selective treatment of unwanted memories associated with psychiatric disorders that is both selective and does not rely on retrieval of the memory. The results further suggest that memory maintenance depends upon the preservation of polymerized actin. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

    The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. DOI: http://dx.doi.org/10.7554/eLife.05361.001 PMID:25866928

  6. Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

    Science.gov (United States)

    Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

    2018-05-10

    The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue

  7. Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure.

    Science.gov (United States)

    Pasakarnis, Laurynas; Frei, Erich; Caussinus, Emmanuel; Affolter, Markus; Brunner, Damian

    2016-11-01

    Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.

  8. Excited hydrogen bonds in the molecular mechanism of muscle contraction.

    Science.gov (United States)

    Bespalova, S V; Tolpygo, K B

    1991-11-21

    The mechanism of muscle contraction is considered. The hydrolysis of an ATP molecule is assumed to produce the excitation of hydrogen bonds A--H...B between electronegative atoms A and B, which are contained in the myosin head and actin filament. This excitation energy epsilon f depends on the interatomic distance AB = R and generates the tractive force f = -delta epsilon f/delta R, that makes atoms AB approach each other. The swing of the myosin head results in macroscopic mutual displacement of actin and myosin polymers. The motion of the actin filament under the action of this force is studied. The conditions under which a considerable portion of the excitation energy converts into the potential tension energy of the actin filament are analysed, and the probability of higher muscle efficiency existence is discussed.

  9. Theory of muscle contraction mechanism with cooperative interaction among crossbridges.

    Science.gov (United States)

    Mitsui, Toshio; Ohshima, Hiroyuki

    2012-01-01

    The power stroke model was criticized and a model was proposed for muscle contraction mechanism (Mitsui, 1999). The proposed model was further developed and calculations based on the model well reproduced major experimental data on the steady filament sliding (Mitsui and Ohshima, 2008) and on the transient phenomena (Mitsui, Takai and Ohshima, 2011). In this review more weight is put on explanation of the basic ideas of the model, especially logical necessity of the model, leaving mathematical details to the above-mentioned papers. A thermodynamic relationship that any models based upon the sliding filament theory should fulfill is derived. The model which fulfills the thermodynamic relationship is constructed on the assumption that a myosin head bound to an actin filament forms a complex with three actin molecules. In shortening muscles, the complex moves along the actin filament changing the partner actin molecules with steps of about 5.5 nm. This process is made possible through cooperative interaction among cross-bridges. The ATP hydrolysis energy is liberated by fraction at each step through chemical reactions between myosin and actin molecules. The cooperativity among crossbridges disappears in length-clamped muscles, in agreement with experimental observations that the cross-bridge produces force independently in the isometric tetanus state. The distance of the head movement per ATP hydrolysis cycle is expected to be about 5.5 nm or a few times of it under the condition of the in vitro single head experiments. Calculation results are surveyed illustrating that they are in good agreement with major experimental observations.

  10. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  11. Changes in myosin S1 orientation and force induced by a temperature increase.

    Science.gov (United States)

    Griffiths, Peter J; Bagni, Maria A; Colombini, Barbara; Amenitsch, Heinz; Bernstorff, Sigrid; Ashley, Christopher C; Cecchi, Giovanni; Ameritsch, Heinz

    2002-04-16

    Force generation in myosin-based motile systems is thought to result from an angular displacement of the myosin subfragment 1 (S1) tail domain with respect to the actin filament axis. In muscle, raised temperature increases the force generated by S1, implying a greater change in tail domain angular displacement. We used time-resolved x-ray diffraction to investigate the structural corollary of this force increase by measuring M3 meridional reflection intensity during sinusoidal length oscillations. This technique allows definition of S1 orientation with respect to the myofilament axis. M3 intensity changes were approximately sinusoid at low temperatures but became increasingly distorted as temperature was elevated, with the formation of a double intensity peak at maximum shortening. This increased distortion could be accounted for by assuming a shift in orientation of the tail domain of actin-bound S1 toward the orientation at which M3 intensity is maximal, which is consistent with a tail domain rotation model of force generation in which the tail approaches a more perpendicular projection from the thin filament axis at higher temperatures. In power stroke simulations, the angle between S1 tail mean position during oscillations and the position at maximum intensity decreased by 4.7 degrees, corresponding to a mean tail displacement toward the perpendicular of 0.73 nm for a temperature-induced force increase of 0.28 P(0) from 4 to 22 degrees C. Our findings suggest that at least 62% of crossbridge compliance is associated with the tail domain.

  12. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-01

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

  14. Leading tip drives soma translocation via forward F-actin flow during neuronal migration.

    Science.gov (United States)

    He, Min; Zhang, Zheng-hong; Guan, Chen-bing; Xia, Di; Yuan, Xiao-bing

    2010-08-11

    Neuronal migration involves coordinated extension of the leading process and translocation of the soma, but the relative contribution of different subcellular regions, including the leading process and cell rear, in driving soma translocation remains unclear. By local manipulation of cytoskeletal components in restricted regions of cultured neurons, we examined the molecular machinery underlying the generation of traction force for soma translocation during neuronal migration. In actively migrating cerebellar granule cells in culture, a growth cone (GC)-like structure at the leading tip exhibits high dynamics, and severing the tip or disrupting its dynamics suppressed soma translocation within minutes. Soma translocation was also suppressed by local disruption of F-actin along the leading process but not at the soma, whereas disrupting microtubules along the leading process or at the soma accelerated soma translocation. Fluorescent speckle microscopy using GFP-alpha-actinin showed that a forward F-actin flow along the leading process correlated with and was required for soma translocation, and such F-actin flow depended on myosin II activity. In migrating neurons, myosin II activity was high at the leading tip but low at the soma, and increasing or decreasing this front-to-rear difference accelerated or impeded soma advance. Thus, the tip of the leading process actively pulls the soma forward during neuronal migration through a myosin II-dependent forward F-actin flow along the leading process.

  15. The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle.

    Science.gov (United States)

    Cooke, Roger

    2011-03-01

    Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.

  16. Phosphorylation of Tropomyosin Extends Cooperative Binding of Myosin Beyond a Single Regulatory Unit

    OpenAIRE

    Rao, Vijay S.; Marongelli, Ellisha N.; Guilford, William H.

    2009-01-01

    Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively ...

  17. Extending the molecular clutch beyond actin-based cell motility

    International Nuclear Information System (INIS)

    Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

    2014-01-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton. (paper)

  18. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    Science.gov (United States)

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K-powered ER/actin network.

    Science.gov (United States)

    Yang, Qinghua; Li, Xiaoyang; Tu, Haitao; Pan, Shen Q

    2017-03-14

    Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium -delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium -delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium -delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation.

  1. Structure and Function of an Actin-Based Filter in the Proximal Axon

    Directory of Open Access Journals (Sweden)

    Varuzhan Balasanyan

    2017-12-01

    Full Text Available Summary: The essential organization of microtubules within neurons has been described; however, less is known about how neuronal actin is arranged and the functional implications of its arrangement. Here, we describe, in live cells, an actin-based structure in the proximal axon that selectively prevents some proteins from entering the axon while allowing the passage of others. Concentrated patches of actin in proximal axons are present shortly after axonal specification in rat and zebrafish neurons imaged live, and they mark positions where anterogradely traveling vesicles carrying dendritic proteins halt and reverse. Patches colocalize with the ARP2/3 complex, and when ARP2/3-mediated nucleation is blocked, a dendritic protein mislocalizes to the axon. Patches are highly dynamic, with few persisting longer than 30 min. In neurons in culture and in vivo, actin appears to form a contiguous, semipermeable barrier, despite its apparently sparse distribution, preventing axonal localization of constitutively active myosin Va but not myosin VI. : Balasanyan et al. find dynamic patches of actin in proximal axons of live neurons, mature and newly differentiated, in culture and in vivo. Patches contribute to a filter that sequesters some proteins within the somatodendritic domain while allowing others to pass into the axon, leading to polarized localization of proteins.

  2. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  3. CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

    Science.gov (United States)

    Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    R. D. Labyntseva,

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Syamaladevi Divya P

    2011-09-01

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

  6. Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain*

    Science.gov (United States)

    Mazerik, Jessica N.; Tyska, Matthew J.

    2012-01-01

    One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts. PMID:22367206

  7. Actin dynamics, architecture, and mechanics in cell motility.

    Science.gov (United States)

    Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

    2014-01-01

    Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

  8. A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

    Science.gov (United States)

    Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

  9. Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

    DEFF Research Database (Denmark)

    Haagensen, L.; Jensen, D.H.; Gesser, Hans

    2008-01-01

    The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyr......The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP...... by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Covalent and non-covalent chemical engineering of actin for biotechnological applications.

    Science.gov (United States)

    Kumar, Saroj; Mansson, Alf

    2017-11-15

    The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-05-01

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

  13. Non-Straub type actin from molluscan catch muscle

    Energy Technology Data Exchange (ETDEWEB)

    Shelud' ko, Nikolay S., E-mail: sheludko@stl.ru; Girich, Ulyana V.; Lazarev, Stanislav S.; Vyatchin, Ilya G.

    2016-05-27

    We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Crenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6 M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization–depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers. -- Highlights: •We developed method of repolymerizable invertebrate smooth muscle actin obtaining. •Our method does not involve use of denaturating agents, which could modify proteins. •Viscosity and polymerization rate of actin, gained that way, is similar to Straub one. •Electron microscopy showed that repolymerized mussel actin is similar to Straub one. •Repolymerized mussel actin has greater ATPase activating capacity, than Straub actin.

  14. INSIGHTS INTO THE MECHANICS OF CYTOKINETIC RING ASSEMBLY USING 3D MODELING.

    Science.gov (United States)

    Bidone, Tamara Carla; Tang, Haosu; Vavylonis, Dimitrios

    During fission yeast cytokinesis, actin filaments nucleated by cortical formin Cdc12 are captured by myosin motors bound to a band of cortical nodes. The myosin motors exert forces that pull nodes together into a contractile ring. Cross-linking interactions help align actin filaments and nodes into a single bundle. Mutations in the myosin motor domain and changes in the concentration of cross-linkers alpha-actinin and fimbrin alter the morphology of the condensing network, leading to clumps, rings or extended meshworks. How the contractile tension developing during ring formation depends on the interplay between network morphology, myosin motor activity, cross-linking and actin filament turnover remains to be elucidated. We addressed this question using a 3D computational model in which semiflexible actin filaments (represented as beads connected by springs) grow from formins, can be captured by myosin in neighboring nodes, and get cross-linked with one another through an attractive interaction. We identify regimes of tension generation between connected nodes under a wide set of conditions regarding myosin dynamics and strength of cross-linking between actin filaments. We find conditions that maximize circumferential tension, correlate them with network morphology and propose experiments to test these predictions. This work addresses "Morphogenesis of soft and living matter" using computational modeling to simulate cytokinetic ring assembly from the key molecular mechanisms of viscoelastic cross-linked actin networks that include active molecular motors.

  15. Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2011-05-01

    The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

  16. Bundling Actin Filaments From Membranes: Some Novel Players

    Directory of Open Access Journals (Sweden)

    Clément eThomas

    2012-08-01

    Full Text Available Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling.

  17. 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

    2010-12-01

    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

  18. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    Science.gov (United States)

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-09

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  19. Mathematical modeling of Myosin induced bistability of Lamellipodial fragments.

    Science.gov (United States)

    Hirsch, S; Manhart, A; Schmeiser, C

    2017-01-01

    For various cell types and for lamellipodial fragments on flat surfaces, externally induced and spontaneous transitions between symmetric nonmoving states and polarized migration have been observed. This behavior is indicative of bistability of the cytoskeleton dynamics. In this work, the Filament Based Lamellipodium Model (FBLM), a two-dimensional, anisotropic, two-phase continuum model for the dynamics of the actin filament network in lamellipodia, is extended by a new description of actin-myosin interaction. For appropriately chosen parameter values, the resulting model has bistable dynamics with stable states showing the qualitative features observed in experiments. This is demonstrated by numerical simulations and by an analysis of a strongly simplified version of the FBLM with rigid filaments and planar lamellipodia at the cell front and rear.

  20. Towards a molecular understanding of the apicomplexan actin motor: on a road to novel targets for malaria remedies?

    Energy Technology Data Exchange (ETDEWEB)

    Kumpula, Esa-Pekka [University of Oulu, PO Box 3000, 90014 Oulu (Finland); Helmholtz Centre for Infection Research, Notkestrasse 85, 22607 Hamburg (Germany); German Electron Synchrotron, Notkestrasse 85, 22607 Hamburg (Germany); Kursula, Inari, E-mail: inari.kursula@helmholtz-hzi.de [University of Oulu, PO Box 3000, 90014 Oulu (Finland); Helmholtz Centre for Infection Research, Notkestrasse 85, 22607 Hamburg (Germany); German Electron Synchrotron, Notkestrasse 85, 22607 Hamburg (Germany); University of Bergen, Jonas Lies vei 91, 5009 Bergen (Norway)

    2015-04-16

    In this review, current structural understanding of the apicomplexan glideosome and actin regulation is described. Apicomplexan parasites are the causative agents of notorious human and animal diseases that give rise to considerable human suffering and economic losses worldwide. The most prominent parasites of this phylum are the malaria-causing Plasmodium species, which are widespread in tropical and subtropical regions, and Toxoplasma gondii, which infects one third of the world’s population. These parasites share a common form of gliding motility which relies on an actin–myosin motor. The components of this motor and the actin-regulatory proteins in Apicomplexa have unique features compared with all other eukaryotes. This, together with the crucial roles of these proteins, makes them attractive targets for structure-based drug design. In recent years, several structures of glideosome components, in particular of actins and actin regulators from apicomplexan parasites, have been determined, which will hopefully soon allow the creation of a complete molecular picture of the parasite actin–myosin motor and its regulatory machinery. Here, current knowledge of the function of this motor is reviewed from a structural perspective.

  1. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing

    Science.gov (United States)

    Fang, Qing; Indzhykulian, Artur A; Mustapha, Mirna; Riordan, Gavin P; Dolan, David F; Friedman, Thomas B; Belyantseva, Inna A; Frolenkov, Gregory I; Camper, Sally A; Bird, Jonathan E

    2015-01-01

    The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture. DOI: http://dx.doi.org/10.7554/eLife.08627.001 PMID:26302205

  2. Biochemical Properties of Soluble and Bound Peroxidases from Artichoke Heads and Leaves

    Directory of Open Access Journals (Sweden)

    Angela Cardinali

    2009-01-01

    Full Text Available Soluble (SP, ionically bound (IBP and covalently bound (CBP peroxidases (POD from artichoke leaves and heads have been characterized for the main biochemical parameters. The three PODs, in both leaves and heads, showed the major apparent catalytic efficiency (vmax,app/Km,app towards ferulic acid, even though, in some cases, they showed higher affinity (Km,app for other substrates. In leaves, SP and IBP showed higher Km,app for ferulic and chlorogenic acids, and CBP for ferulic and caffeic acids. In heads, SP showed higher Km,app for chlorogenic acid, IBP for caffeic and ferulic acids, and CBP for ferulic acid. It was shown that pH optimum for PODs ranged between 5.0 and 6.0 in leaves. In heads, pH optimum for SP and IBP was 5.5, while CBP presented a very low activity in a wide pH range. All PODs showed high thermal stability but different ability to regenerate: the bound forms were more able to regenerate than the soluble one. The results obtained show that (i CBP from heads is able to work under very different cellular conditions, (ii all PODs, in both tissues, have a high apparent catalytic efficiency for ferulic acid, which could explain the effective involvement of POD in lignin biosynthesis, (iii in heads, high Km,app of SP for chlorogenic acid, particularly abundant in artichoke, could justify the possible involvement of PODs in browning mechanism, and (iv in heat-processed artichoke, the ability of PODs to regenerate could contribute to oxidation and loss of product quality.

  3. Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

    Science.gov (United States)

    Green, Judith L; Wall, Richard J; Vahokoski, Juha; Yusuf, Noor A; Ridzuan, Mohd A Mohd; Stanway, Rebecca R; Stock, Jessica; Knuepfer, Ellen; Brady, Declan; Martin, Stephen R; Howell, Steven A; Pires, Isa P; Moon, Robert W; Molloy, Justin E; Kursula, Inari; Tewari, Rita; Holder, Anthony A

    2017-10-27

    Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use

    Science.gov (United States)

    Young, Erica J.; Blouin, Ashley M.; Briggs, Sherri B.; Sillivan, Stephanie E.; Lin, Li; Cameron, Michael D.; Rumbaugh, Gavin; Miller, Courtney A.

    2015-01-01

    Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD) and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin II (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin, a small molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, post-consolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebbistatin was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted twenty-four hours after a single systemic injection of Blebbistatin delivered in the home cage. Together, these results argue for the further development of small molecule inhibitors of nonmuscle myosin II as potential therapeutics for the prevention of SUD relapse triggered by drug associations. PMID:26239291

  5. Myosin XI-Dependent Formation of Tubular Structures from Endoplasmic Reticulum Isolated from Tobacco Cultured BY-2 Cells1[W][OA

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2011-01-01

    The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER. PMID:21427277

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

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

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

  7. Regulation of Contraction by the Thick Filaments in Skeletal Muscle.

    Science.gov (United States)

    Irving, Malcolm

    2017-12-19

    Contraction of skeletal muscle cells is initiated by a well-known signaling pathway. An action potential in a motor nerve triggers an action potential in a muscle cell membrane, a transient increase of intracellular calcium concentration, binding of calcium to troponin in the actin-containing thin filaments, and a structural change in the thin filaments that allows myosin motors from the thick filaments to bind to actin and generate force. This calcium/thin filament mediated pathway provides the "START" signal for contraction, but it is argued that the functional response of the muscle cell, including the speed of its contraction and relaxation, adaptation to the external load, and the metabolic cost of contraction is largely determined by additional mechanisms. This review considers the role of the thick filaments in those mechanisms, and puts forward a paradigm for the control of contraction in skeletal muscle in which both the thick and thin filaments have a regulatory function. The OFF state of the thick filament is characterized by helical packing of most of the myosin head or motor domains on the thick filament surface in a conformation that makes them unavailable for actin binding or ATP hydrolysis, although a small fraction of the myosin heads are constitutively ON. The availability of the majority fraction of the myosin heads for contraction is controlled in part by the external load on the muscle, so that these heads only attach to actin and hydrolyze ATP when they are required. This phenomenon seems to be the major determinant of the well-known force-velocity relationship of muscle, and controls the metabolic cost of contraction. The regulatory state of the thick filament also seems to control the dynamics of both muscle activation and relaxation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. The actin multigene family of Paramecium tetraurelia

    Directory of Open Access Journals (Sweden)

    Wagner Erika

    2007-03-01

    Full Text Available Abstract Background A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. Results The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic

  9. Distortion of the Actin A-Triad Results in Contractile Disinhibition and Cardiomyopathy

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    Meera C. Viswanathan

    2017-09-01

    Full Text Available Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder.

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

    NARCIS (Netherlands)

    Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

    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.

  11. Decidable and undecidable arithmetic functions in actin filament networks

    Science.gov (United States)

    Schumann, Andrew

    2018-01-01

    The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

  12. Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

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    Sundaravadivel Balasubramanian

    2010-07-01

    Full Text Available The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring

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

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    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.

  14. Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

    Science.gov (United States)

    Kim, Kyoungtae; Keller, Thomas C S

    2002-01-07

    Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

  15. 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.

  16. CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI.

    Science.gov (United States)

    Salles, Felipe T; Andrade, Leonardo R; Tanda, Soichi; Grati, M'hamed; Plona, Kathleen L; Gagnon, Leona H; Johnson, Kenneth R; Kachar, Bechara; Berryman, Mark A

    2014-01-01

    Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. © Published 2013 Wiley Periodicals, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

  17. Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

    Science.gov (United States)

    Calábria, Luciana Karen; Peixoto, Pablo Marco Veras; Passos Lima, Andreia Barcelos; Peixoto, Leonardo Gomes; de Moraes, Viviane Rodrigues Alves; Teixeira, Renata Roland; Dos Santos, Claudia Tavares; E Silva, Letícia Oliveira; da Silva, Maria de Fátima Rodrigues; dos Santos, Ana Alice Diniz; Garcia-Cairasco, Norberto; Martins, Antônio Roberto; Espreafico, Enilza Maria; Espindola, Foued Salmen

    2011-09-01

    Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis.

    Science.gov (United States)

    Buisson, Nicolas; Sirour, Cathy; Moreau, Nicole; Denker, Elsa; Le Bouffant, Ronan; Goullancourt, Aline; Darribère, Thierry; Bello, Valérie

    2014-12-01

    Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells. © 2014. Published by The Company of Biologists Ltd.

  19. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

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    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  20. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    International Nuclear Information System (INIS)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-01-01

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity

  1. Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

    Directory of Open Access Journals (Sweden)

    Felipe O Bendezú

    Full Text Available The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

  2. Actin is an essential component of plant gravitropic signaling pathways

    Science.gov (United States)

    Braun, Markus; Hauslage, Jens; Limbach, Christoph

    2003-08-01

    A role of the actin cytoskeleton in the different phases of gravitropism in higher plant organs seems obvious, but experimental evidence is still inconclusive and contradictory. In gravitropically tip-growing rhizoids and protonemata, however, it is well documented that actin is an essential component of the tip-growth machinery and is involved either in the cellular mechanisms that lead to gravity sensing and in the processes of the graviresponses that result in the reorientation of the growth direction. All these processes depend on a complexly organized and highly dynamic organization of actin filaments whose diverse functions are coordinated by numerous associated proteins. Actin filaments and myosins mediate the transport of secretory vehicles to the growing tip and precisely control the delivery of cell wall material. In addition, both cell types use a very efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. The studies presented in this paper provide evidence for the essential role of actin in plant gravity sensing and the gravitropic responses. A unique actin-organizing center exists in the tip of characean rhizoids and protonemata which is associated with and dynamically regulated by a specific set of actin-dynamizing proteins. It is concluded that this highly dynamic apical actin array is an essential prerequisite for gravity sensing and gravity-oriented tip growth.

  3. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion.

    Science.gov (United States)

    Scott, Kevin G-E; Meddings, Jonathon B; Kirk, David R; Lees-Miller, Susan P; Buret, André G

    2002-10-01

    Giardiasis causes malabsorptive diarrhea, and symptoms can be present in the absence of any significant morphologic injury to the intestinal mucosa. The effects of giardiasis on epithelial permeability in vivo remain unknown, and the role of T cells and myosin light chain kinase (MLCK) in altered intestinal barrier function is unclear. This study was conducted to determine whether Giardia spp. alters intestinal permeability in vivo, to assess whether these abnormalities are dependent on T cells, and to assess the role of MLCK in altered epithelial barrier function. Immunocompetent and isogenic athymic mice were inoculated with axenic Giardia muris trophozoites or sterile vehicle (control), then assessed for trophozoite colonization and gastrointestinal permeability. Mechanistic studies using nontransformed human duodenal epithelial monolayers (SCBN) determined the effects of Giardia on myosin light chain (MLC) phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, cytoskeletal F-actin, tight junctional zonula occludens-1 (ZO-1), and MLCK. Giardia infection caused a significant increase in small intestinal, but not gastric or colonic, permeability that correlated with trophozoite colonization in both immunocompetent and athymic mice. In vitro, Giardia increased permeability and phosphorylation of MLC and reorganized F-actin and ZO-1. These alterations were abolished with an MLCK inhibitor. Disruption of small intestinal barrier function is T cell independent, disappears on parasite clearance, and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1 in an MLCK-dependent fashion.

  4. Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons.

    Science.gov (United States)

    Perrin, Laura; Roudeau, Stéphane; Carmona, Asuncion; Domart, Florelle; Petersen, Jennifer D; Bohic, Sylvain; Yang, Yang; Cloetens, Peter; Ortega, Richard

    2017-07-19

    Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons. We found that zinc is distributed all along the dendrites while copper is mainly pinpointed within the spines. In spines, zinc content is higher within the spine head while copper is higher within the spine neck. Such specific distributions suggested metal interactions with cytoskeleton proteins. Zinc supplementation induced the increase of β-tubulin content in dendrites. Copper supplementation impaired the β-tubulin and F-actin networks. Copper chelation resulted in the decrease of F-actin content in dendrites, drastically reducing the number of F-actin protrusions. These results indicate that zinc is involved in microtubule stability whereas copper is essential for actin-dependent stability of dendritic spines, although copper excess can impair the dendritic cytoskeleton.

  5. Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

    Science.gov (United States)

    Baxley, Tamatha; Johnson, Dylan; Pinto, Jose R; Chalovich, Joseph M

    2017-06-13

    Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca 2+ and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca 2+ concentration stabilizes the most active state (M state) of the actin-tropomyosin-troponin complex (regulated actin). Ca 2+ binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca 2+ concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca 2+ sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca 2+ concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca 2+ concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca 2+ . Because Ca 2+ gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

  6. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

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

    International Nuclear Information System (INIS)

    Maruta, H.; Korn, E.D.

    1981-01-01

    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 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

  8. Use of actin-bound adenosine 5'-diphosphate as a method to determine the specific 32P-radioactivity of the gamma-phosphoryl group of adenosine 5'-triphosphate in a highly compartmentalized cell, the platelet

    International Nuclear Information System (INIS)

    Verhoeven, A.J.; Cook, C.A.; Holmsen, H.

    1988-01-01

    Determination of the specific 32 P-radioactivity of cytoplasmic ATP in 32 P-Pi-labeled platelets is complicated by the presence of a large pool of metabolically inactive, granule-stored nucleotides. Moreover, our data show that the specific 32 P-radioactivity of cytoplasmic ATP is severely underestimated when determined in platelets after the complete secretion of granule-stored nucleotides, possibly due to isotopic dilution with granule-stored phosphate. As F-actin-bound ADP is ethanol-insoluble, this pool can be readily separated from the other nucleotide pools in platelets. Here we show that the specific 32 P-radioactivity of F-actin-bound ADP accurately reflects that of the gamma-phosphoryl group of cytoplasmic ATP. During uptake of 32 P-Pi by human platelets the specific 32 P-radioactivity of F-actin-bound ADP equals that of the monoester phosphates of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which are in metabolic equilibrium with cytoplasmic ATP. Therefore, this method enables the determination of the specific 32 P-radioactivity of the gamma-phosphoryl group of cytoplasmic ATP in platelets even under short-term labeling conditions

  9. Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle.

    Science.gov (United States)

    Kampourakis, Thomas; Ponnam, Saraswathi; Irving, Malcolm

    2018-04-01

    Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

    Science.gov (United States)

    Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

    2018-03-01

    Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

  11. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    International Nuclear Information System (INIS)

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-01-01

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  12. Using optical tweezers to relate the chemical and mechanical cross-bridge cycles.

    Science.gov (United States)

    Steffen, Walter; Sleep, John

    2004-12-29

    In most current models of muscle contraction there are two translational steps, the working stroke, whereby an attached myosin cross-bridge moves relative to the actin filament, and the repriming step, in which the cross-bridge returns to its original orientation. The development of single molecule methods has allowed a more detailed investigation of the relationship of these mechanical steps to the underlying biochemistry. In the normal adenosine triphosphate cycle, myosin.adenosine diphosphate.phosphate (M.ADP.Pi) binds to actin and moves it by ca. 5 nm on average before the formation of the end product, the rigor actomyosin state. All the other product-like intermediate states tested were found to give no net movement indicating that M.ADP.Pi alone binds in a pre-force state. Myosin states with bound, unhydrolysed nucleoside triphosphates also give no net movement, indicating that these must also bind in a post-force conformation and that the repriming, post- to pre-transition during the forward cycle must take place while the myosin is dissociated from actin. These observations fit in well with the structural model in which the working stroke is aligned to the opening of the switch 2 element of the ATPase site.

  13. The 5’cap of Tobacco Mosaic Virus (TMV) is required for virion attachment to the actin/ER network during early infection

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Tilsner, Jens; Bell, Karen

    to the motile cortical actin/ER network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actindependent RNA movement. The 5’ methylguanosine TMV cap was shown to be required for vRNA anchoring to the ER. TMV vRNA lacking the 5’cap failed to form granules...... the fluorescent vRNA pool nor co-injected GFP left the injected trichome, indicating that the synthesis of unlabelled progeny viral (v)RNA is required to initiate cell-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored...... on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome....

  14. Cytoskeletal dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    I worked with reconstitutted contractile acto-myosin systems containing mainly actin, actin cross-linkers and myosin motors. Contractility and rheology of such systems was studied using confocal microscopy and rheology.......I worked with reconstitutted contractile acto-myosin systems containing mainly actin, actin cross-linkers and myosin motors. Contractility and rheology of such systems was studied using confocal microscopy and rheology....

  15. Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation

    Directory of Open Access Journals (Sweden)

    Simone Mortal

    2017-12-01

    Full Text Available During neurite development, Actin Waves (AWs emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC. Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration.

  16. Stochastic Severing of Actin Filaments by Actin Depolymerizing Factor/Cofilin Controls the Emergence of a Steady Dynamical Regime

    Science.gov (United States)

    Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis

    2008-01-01

    Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447

  17. Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

    Science.gov (United States)

    Marshall Mccall, Patrick

    Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non

  18. Unconventional Imaging Methods to Capture Transient Structures during Actomyosin Interaction

    Directory of Open Access Journals (Sweden)

    Eisaku Katayama

    2018-05-01

    Full Text Available Half a century has passed since the cross-bridge structure was recognized as the molecular machine that generates muscle tension. Despite various approaches by a number of scientists, information on the structural changes in the myosin heads, particularly its transient configurations, remains scant even now, in part because of their small size and rapid stochastic movements during the power stroke. Though progress in cryo-electron microscopy is eagerly awaited as the ultimate means to elucidate structural details, the introduction of some unconventional methods that provide high-contrast raw images of the target protein assemblies is quite useful, if available, to break the current impasse. Quick-freeze deep–etch–replica electron microscopy coupled with dedicated image analysis procedures, and high-speed atomic-force microscopy are two such candidates. We have applied the former to visualize actin-associated myosin heads under in vitro motility assay conditions, and found that they take novel configurations similar to the SH1–SH2-crosslinked myosin that we characterized recently. By incorporating biochemical and biophysical results, we have revised the cross-bridge mechanism to involve the new conformer as an important main player. The latter “microscopy” is unique and advantageous enabling continuous observation of various protein assemblies as they function. Direct observation of myosin-V’s movement along actin filaments revealed several unexpected behaviors such as foot-stomping of the leading head and unwinding of the coiled-coil tail. The potential contribution of these methods with intermediate spatial resolution is discussed.

  19. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.

    1986-01-01

    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  20. MicroRNA-363 targets myosin 1B to reduce cellular migration in head and neck cancer

    International Nuclear Information System (INIS)

    Chapman, Bhavana V.; Wald, Abigail I.; Akhtar, Parvez; Munko, Ana C.; Xu, Jingjing; Gibson, Sandra P.; Grandis, Jennifer R.; Ferris, Robert L.; Khan, Saleem A.

    2015-01-01

    Squamous cell carcinoma of the head and neck (SCCHN) remains a prevalent and devastating disease. Recently, there has been an increase in SCCHN cases that are associated with high-risk human papillomavirus (HPV) infection. The clinical characteristics of HPV-positive and HPV-negative SCCHN are known to be different but their molecular features are only recently beginning to emerge. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs that are likely to play significant roles in cancer initiation and progression where they may act as oncogenes or tumor suppressors. Previous studies in our laboratory showed that miR-363 is overexpressed in HPV-positive compared to HPV-negative SCCHN cell lines, and the HPV type 16-E6 oncoprotein upregulates miR-363 in SCCHN cell lines. However, the functional role of miR-363 in SCCHN in the context of HPV infection remains to be elucidated. We analyzed miR-363 levels in SCCHN tumors with known HPV-status from The Cancer Genome Atlas (TCGA) and an independent cohort from our institution. Cell migration studies were conducted following the overexpression of miR-363 in HPV-negative cell lines. Bioinformatic tools and a luciferase reporter assay were utilized to confirm that miR-363 targets the 3’-UTR of myosin 1B (MYO1B). MYO1B mRNA and protein expression levels were evaluated following miR-363 overexpression in HPV-negative SCCHN cell lines. Small interfering RNA (siRNA) knockdown of MYO1B was performed to assess the phenotypic implication of reduced MYO1B expression in SCCHN cell lines. MiR-363 was found to be overexpressed in HPV-16-positive compared to the HPV-negative SCCHN tumors. Luciferase reporter assays performed in HPV-negative JHU028 cells confirmed that miR-363 targets one of its two potential binding sites in the 3’UTR of MYO1B. MYO1B mRNA and protein levels were reduced upon miR-363 overexpression in four HPV-negative SCCHN cell lines. Increased miR-363 expression or siRNA knockdown of MYO1B expression reduced

  1. Actin, actin-binding proteins, and actin-related proteins in the nucleus.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

    2016-04-01

    Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

  2. Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells.

    Science.gov (United States)

    Glenn, Honor L; Jacobson, Bruce S

    2003-08-01

    The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility. Copyright 2003 Wiley-Liss, Inc.

  3. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    Science.gov (United States)

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  4. Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

    Science.gov (United States)

    Ravenscroft, Gianina; Jackaman, Connie; Sewry, Caroline A.; McNamara, Elyshia; Squire, Sarah E.; Potter, Allyson C.; Papadimitriou, John; Griffiths, Lisa M.; Bakker, Anthony J.; Davies, Kay E.; Laing, Nigel G.; Nowak, Kristen J.

    2011-01-01

    Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations. PMID:22174871

  5. Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers

    Directory of Open Access Journals (Sweden)

    Yaming Jiu

    2015-06-01

    Full Text Available The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis.

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

    International Nuclear Information System (INIS)

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-01-01

    Highlights: ► Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. ► Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. ► It is a widely believed that MYL2 is a poor substrate for smMLCK. ► In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. ► 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 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 max and K 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 kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-16

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

  8. Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

    Science.gov (United States)

    Badirou, Idinath; Pan, Jiajia; Legrand, Céline; Wang, Aibing; Lordier, Larissa; Boukour, Siham; Roy, Anita; Vainchenker, William; Chang, Yunhua

    2014-10-16

    Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

  9. Expression of smooth muscle and non-muscle myosin heavy chain isoforms in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Rovner, A.S.; Murphy, R.A.; Owens, G.K.

    1986-01-01

    Immunocytochemical studies of cultured smooth muscle cells (SMCs) have disagreed on the nature of myosin expression. This investigation was undertaken to test for the presence of heterogeneous myosin heavy chain (MHC) isoforms in cell culture as a possible explanation for these results. Previously, Rovner et al. detected two MHCs in intact smooth muscles which differed in molecular weight by ca. 4000 daltons (SM1 and SM2) using a 3-4% acrylamide gradient SDS gel system. When sub-confluent primary cultures of rat aorta SMCs were assayed by this system, SM1 and SM2 were seen, along with large amounts of a third, unique MHC, NM, which closely resembled the MHC from human platelet in size and antigenicity. Data from 35 S-methionine autoradiograms showed that the log growth phase SMC cultures were producing almost exclusively NM, but the growth arrest, post-confluent cultures synthesized increased relative amounts of the SM MHC forms and contained comparable amounts of SM1, SM2, and NM. The same patterns of MHC synthesis were seen in sub-passaged SMCs. The expression of the SM-specific forms of myosin in quiescent, post-confluent cultures parallels that of smooth muscle actin suggesting that density induced growth arrest promotes cytodifferentiation in cultured vascular SMCs

  10. Analysis of myofibrillar proteins and transcripts in adult skeletal muscles of the American lobster Homarus americanus: variable expression of myosins, actin and troponins in fast, slow-twitch and slow-tonic fibres.

    Science.gov (United States)

    Medler, Scott; Mykles, Donald L

    2003-10-01

    Skeletal muscles are diverse in their contractile properties, with many of these differences being directly related to the assemblages of myofibrillar isoforms characteristic of different fibers. Crustacean muscles are similar to other muscles in this respect, although the majority of information about differences in muscle organization comes from vertebrate species. In the present study, we examined the correlation between myofibrillar protein isoforms and the patterns of myofibrillar gene expression in fast, slow-phasic (S(1)) and slow-tonic (S(2)) fibers of the American lobster Homarus americanus. SDS-PAGE and western blotting were used to identify isoform assemblages of myosin heavy chain (MHC), P75, troponin T (TnT) and troponin I (TnI). RT-PCR was used to monitor expression of fast and slow (S(1)) MHC, P75 and actin in different fiber types, and the MHC and actin levels were quantified by real-time PCR. Fast and slow fibers from the claw closers predominantly expressed fast and S(1) MHC, respectively, but also lower levels of the alternate MHC. By contrast, fast fibers from the deep abdominal muscle expressed fast MHC exclusively. In addition, slow muscles expressed significantly higher levels of actin than fast fibers. A distal bundle of fibers in the cutter claw closer muscle was found to be composed of a mixture of S(1) and S(2) fibers, many of which possessed a mixture of S(1) and S(2) MHC isoforms. This pattern supports the idea that S(1) and S(2) fibers represent extremes in a continuum of slow muscle phenotype. Overall, these patterns demonstrate that crustacean skeletal muscles cannot be strictly categorized into discrete fiber types, but a muscle's properties probably represent a point on a continuum of fiber types. This trend may result from differences in innervation pattern, as each muscle is controlled by a unique combination of phasic, tonic or both phasic and tonic motor nerves. In this respect, future studies examining how muscle phenotype

  11. Multiple essential functions of Plasmodium falciparum actin-1 during malaria blood-stage development.

    Science.gov (United States)

    Das, Sujaan; Lemgruber, Leandro; Tay, Chwen L; Baum, Jake; Meissner, Markus

    2017-08-15

    The phylum Apicomplexa includes intracellular parasites causing immense global disease burden, the deadliest of them being the human malaria parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The cytoskeletal protein actin is well conserved within apicomplexans but divergent from mammalian actins, and was primarily reported to function during host cell invasion. However, novel invasion mechanisms have been described for several apicomplexans, and specific functions of the acto-myosin system are being reinvestigated. Of the two actin genes in P. falciparum, actin-1 (pfact1) is ubiquitously expressed in all life-cycle stages and is thought to be required for erythrocyte invasion, although its functions during parasite development are unknown, and definitive in vivo characterisation during invasion is lacking. Here we have used a conditional Cre-lox system to investigate the functions of PfACT1 during P. falciparum blood-stage development and host cell invasion. We demonstrate that PfACT1 is crucially required for segregation of the plastid-like organelle, the apicoplast, and for efficient daughter cell separation during the final stages of cytokinesis. Surprisingly, we observe that egress from the host cell is not an actin-dependent process. Finally, we show that parasites lacking PfACT1 are capable of microneme secretion, attachment and formation of a junction with the erythrocyte, but are incapable of host cell invasion. This study provides important mechanistic insights into the definitive essential functions of PfACT1 in P. falciparum, which are not only of biological interest, but owing to functional divergence from mammalian actins, could also form the basis for the development of novel therapeutics against apicomplexans.

  12. Addition of electrophilic lipids to actin alters filament structure

    International Nuclear Information System (INIS)

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.; Terron, Maria C.; Llorca, Oscar; Perez-Sala, Dolores

    2006-01-01

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) and PGA 1 in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA 1 and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ 2 or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ 2 at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles

  13. 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.

  14. Chemotaxis and Actin Oscillations

    Science.gov (United States)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  15. Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

    Science.gov (United States)

    Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

    2010-01-01

    The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

  16. Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers.

    Science.gov (United States)

    Jiu, Yaming; Lehtimäki, Jaakko; Tojkander, Sari; Cheng, Fang; Jäälinoja, Harri; Liu, Xiaonan; Varjosalo, Markku; Eriksson, John E; Lappalainen, Pekka

    2015-06-16

    The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Poorly Understood Aspects of Striated Muscle Contraction

    Directory of Open Access Journals (Sweden)

    Alf Månsson

    2015-01-01

    Full Text Available Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP. Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.

  18. Coupling of lever arm swing and biased Brownian motion in actomyosin.

    Directory of Open Access Journals (Sweden)

    Qing-Miao Nie

    2014-04-01

    Full Text Available An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.

  19. Coupling of lever arm swing and biased Brownian motion in actomyosin.

    Science.gov (United States)

    Nie, Qing-Miao; Togashi, Akio; Sasaki, Takeshi N; Takano, Mitsunori; Sasai, Masaki; Terada, Tomoki P

    2014-04-01

    An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (Pi) and ADP weakly binds to actin and that after releasing Pi and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.

  20. [Cleavage time for a hydrogen bond under a load].

    Science.gov (United States)

    Bespalov, S V; Tolpygo, K B

    1993-01-01

    Statistics of the hydrogen bond formation and break in a bundle of actin and myosin filaments realizing the attractive force in the sarcomere of a muscle is studied. Purely mechanical problem of the attractive-force formation and motion of myosin heads and action globules under their action is supplemented by accounting for the irreversible processes: 1. Thermal de-excitation of the latter in the chain of hydrogen bond during the elementary act of the ATP energy use resulting in fixing the extended actin filament. 2. Break of the hydrogen bonds, realizing this fixing, due to thermal fluctuations for the time tau. The average life-time turns out to be the order of time necessary for the movement of z-membrane sarcomere for the value of action filament extension delta 1, which is necessary for the process of muscle contraction to be continued.

  1. Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells

    Science.gov (United States)

    Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Changyaleket, Benjarat; Whitlon, Donna S.; Mugnaini, Enrico; Bartles, James R.

    2010-01-01

    Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca2+-resistant fashion, bound actin monomer via a WASP homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53 and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5- bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in a variety of mechanosensory and chemosensory cells. PMID:15190118

  2. Myosin light chain kinase knockout improves gut barrier function and confers a survival advantage in polymicrobial sepsis.

    Science.gov (United States)

    Lorentz, C Adam; Liang, Zhe; Meng, Mei; Chen, Ching-Wen; Yoseph, Benyam P; Breed, Elise R; Mittal, Rohit; Klingensmith, Nathan J; Farris, Alton B; Burd, Eileen M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

    2017-06-07

    Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK -/- and wild type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK -/- mice (95% vs. 24%, p<0.0001). Intestinal permeability increased in septic WT mice compared to unmanipulated mice. In contrast, permeability in septic MLCK -/- mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK -/- mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8, 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK -/- mice; however, survival was similar between septic MLCK -/- mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.

  3. Histones bundle F-actin filaments and affect actin structure.

    Science.gov (United States)

    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

    Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  4. Azidoblebbistatin, a photoreactive myosin inhibitor

    Science.gov (United States)

    Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András

    2012-01-01

    Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. PMID:22647605

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

    Directory of Open Access Journals (Sweden)

    Karen A. Newell-Litwa

    2015-12-01

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

  6. Histones bundle F-actin filaments and affect actin structure.

    Directory of Open Access Journals (Sweden)

    Edna Blotnick

    Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  7. Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+](i and force suppression in forskolin-pretreated porcine coronary arteries.

    Directory of Open Access Journals (Sweden)

    Kyle M Hocking

    Full Text Available Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+]i and phosphorylation of myosin light chains (MLC. However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

  8. Crosstalk between Rac1-mediated actin regulation and ROS production.

    Science.gov (United States)

    Acevedo, Alejandro; González-Billault, Christian

    2018-02-20

    The small RhoGTPase Rac1 is implicated in a variety of events related to actin cytoskeleton rearrangement. Remarkably, another event that is completely different from those related to actin regulation has the same relevance; the Rac1-mediated production of reactive oxygen species (ROS) through NADPH oxidases (NOX). Each outcome involves different Rac1 downstream effectors; on one hand, events related to the actin cytoskeleton require Rac1 to bind to WAVEs proteins and PAKs that ultimately promote actin branching and turnover, on the other, NOX-derived ROS production demands active Rac1 to be bound to a cytosolic activator of NOX. How Rac1-mediated signaling ends up promoting actin-related events, NOX-derived ROS, or both is poorly understood. Rac1 regulators, including scaffold proteins, are known to exert tight control over its functions. Hence, evidence of Rac1 regulatory events leading to both actin remodeling and NOX-mediated ROS generation are discussed. Moreover, cellular functions linked to physiological and pathological conditions that exhibit crosstalk between Rac1 outcomes are analyzed, while plausible roles in neuronal functions (and dysfunctions) are highlighted. Together, discussed evidence shed light on cellular mechanisms which requires Rac1 to direct either actin- and/or ROS-related events, helping to understand crucial roles of Rac1 dual functionality. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Interactions between globular proteins and F-actin in isotonic saline solution.

    Science.gov (United States)

    Lakatos, S; Minton, A P

    1991-10-05

    Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.

  10. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  11. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    International Nuclear Information System (INIS)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    2011-01-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  12. Elastic coupling of nascent apCAM adhesions to flowing actin networks.

    Science.gov (United States)

    Mejean, Cecile O; Schaefer, Andrew W; Buck, Kenneth B; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W; Dufresne, Eric R; Forscher, Paul

    2013-01-01

    Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

  13. Elastic coupling of nascent apCAM adhesions to flowing actin networks.

    Directory of Open Access Journals (Sweden)

    Cecile O Mejean

    Full Text Available Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

  14. Robust mechanobiological behavior emerges in heterogeneous myosin systems

    Science.gov (United States)

    Egan, Paul F.; Moore, Jeffrey R.; Ehrlicher, Allen J.; Weitz, David A.; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

    2017-09-01

    Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

  15. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    Science.gov (United States)

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

  16. A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs.

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R; Shuster, Charles B

    2006-09-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

  17. A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

    2006-01-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

  18. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  19. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven

    2014-01-01

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  20. Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

    Science.gov (United States)

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J K; Schubert, Dirk W; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

  1. Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts.

    Science.gov (United States)

    Hu, Guiqing; Taylor, Dianne W; Liu, Jun; Taylor, Kenneth A

    2018-03-01

    Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

    Science.gov (United States)

    Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

    2005-10-01

    From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

  3. Actinic keratosis

    Science.gov (United States)

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... Actinic keratosis is caused by exposure to sunlight. You are more likely to develop it if you: Have fair ...

  4. Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

    Science.gov (United States)

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

    2012-01-01

    Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

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

    International Nuclear Information System (INIS)

    Okada, H.; Souza, I.T.T.

    1990-01-01

    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)

  6. Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11)

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Wijk, E. van; Bischoff, A.M.L.C.; Krieger, E.; Huygen, P.L.M.; Pennings, R.J.E.; Brunner, H.G.; Cremers, C.W.R.J.; Cremers, F.P.M.; Kremer, J.M.J.

    2004-01-01

    Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant

  7. Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11).

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Wijk, E. van; Bischoff, A.M.L.C.; Krieger, E.; Huygen, P.L.M.; Pennings, R.J.E.; Brunner, H.G.; Cremers, C.W.R.J.; Cremers, F.P.M.; Kremer, J.M.J.

    2004-01-01

    Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant

  8. Myosin II dynamics are regulated by tension in intercalating cells.

    Science.gov (United States)

    Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

    2009-11-01

    Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

  9. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    OpenAIRE

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic bindin...

  10. LL-37 induces polymerization and bundling of actin and affects actin structure.

    Directory of Open Access Journals (Sweden)

    Asaf Sol

    Full Text Available Actin exists as a monomer (G-actin which can be polymerized to filaments F-actin that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl(2 or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.

  11. Characterization of myosin light chain in shrimp hemocytic phagocytosis.

    Science.gov (United States)

    Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

    2010-11-01

    Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

  12. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

    Science.gov (United States)

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-12-30

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

  13. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    Science.gov (United States)

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

    2016-04-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

  14. Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

    International Nuclear Information System (INIS)

    Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar; Illenberger, Susanne; Preuss, Ute

    2005-01-01

    Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis

  15. New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

    Science.gov (United States)

    Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

    2006-04-01

    SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

  16. Topology of interaction between titin and myosin thick filaments.

    Science.gov (United States)

    Kellermayer, Miklós; Sziklai, Dominik; Papp, Zsombor; Decker, Brennan; Lakatos, Eszter; Mártonfalvi, Zsolt

    2018-05-05

    Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the A-band titin is associated with the myosin thick filament. It has been speculated that titin may serve as a blueprint for thick-filament formation due to the super-repeat structure of its A-band domains. Accordingly, titin might provide a template that determines the length and structural periodicity of the thick filament. Here we tested the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ratios (300 myosins per 12 titins) in buffers of different ionic strength (KCl concentration range 100-300 mM). The topology of the filamentous complexes was investigated with atomic force microscopy. We found that the samples contained distinct, segregated populations of titin molecules and myosin thick filaments. We were unable to identify complexes in which myosin molecules were regularly associated to either mono- or oligomeric titin in either relaxed or stretched states of the titin filaments. Thus, the electrostatically driven self-association is stronger in both myosin and titin than their binding to each other, and it is unlikely that titin functions as a geometrical template for thick-filament formation. However, when allowed to equilibrate configurationally, long myosin thick filaments appeared with titin oligomers attached to their surface. The titin meshwork formed on the thick-filament surface may play a role in controlling thick-filament length by regulating the structural dynamics of myosin molecules and placing a mechanical limit on the filament length. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. The unique organization of filamentous actin in the medullary canal of the medulla oblongata.

    Science.gov (United States)

    Tan, Bai-Hong; Guo, Chun-Yan; Xiong, Tian-Qing; Chen, Ling-Meng; Li, Yan-Chao

    2017-04-01

    In the central canal, F-actin is predominantly localized in the apical region, forming a ring-like structure around the circumference of the lumen. However, an exception is found in the medulla oblongata, where the apical F-actin becomes interrupted in the ventral aspect of the canal. To clarify the precise localization of F-actin, the fluorescence signals for F-actin were converted to the peroxidase/DAB reaction products in this study by a phalloidin-based ultrastructural technique, which demonstrated that F-actin is located mainly in the microvilli and terminal webs in the ependymocytes. It is because the ventrally oriented ependymocytes do not possess well-developed microvilli or terminal web that led to a discontinuous labeling of F-actin in the medullary canal. Since spinal motions can change the shape and size of the central canal, we next examined the cytoskeletons in the medullary canal in both rats and monkeys, because these two kinds of animals show different kinematics at the atlanto-occipital articulation. Our results first demonstrated that the apical F-actin in the medullary canal is differently organized in the animals with different head-neck kinemics, which suggests that the mechanic stretching of spinal motions is capable of inducing F-actin reorganization and the subsequent cell-shape changes in the central canal. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Interaction between cardiac myosin-binding protein C and formin Fhod3.

    Science.gov (United States)

    Matsuyama, Sho; Kage, Yohko; Fujimoto, Noriko; Ushijima, Tomoki; Tsuruda, Toshihiro; Kitamura, Kazuo; Shiose, Akira; Asada, Yujiro; Sumimoto, Hideki; Takeya, Ryu

    2018-05-08

    Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

  19. Myosin 1g Contributes to CD44 Adhesion Protein and Lipid Rafts Recycling and Controls CD44 Capping and Cell Migration in B Lymphocytes

    Directory of Open Access Journals (Sweden)

    Orestes López-Ortega

    2017-12-01

    Full Text Available Cell migration and adhesion are critical for immune system function and involve many proteins, which must be continuously transported and recycled in the cell. Recycling of adhesion molecules requires the participation of several proteins, including actin, tubulin, and GTPases, and of membrane components such as sphingolipids and cholesterol. However, roles of actin motor proteins in adhesion molecule recycling are poorly understood. In this study, we identified myosin 1g as one of the important motor proteins that drives recycling of the adhesion protein CD44 in B lymphocytes. We demonstrate that the lack of Myo1g decreases the cell-surface levels of CD44 and of the lipid raft surrogate GM1. In cells depleted of Myo1g, the recycling of CD44 was delayed, the delay seems to be caused at the level of formation of recycling complex and entry into recycling endosomes. Moreover, a defective lipid raft recycling in Myo1g-deficient cells had an impact both on the capping of CD44 and on cell migration. Both processes required the transportation of lipid rafts to the cell surface to deliver signaling components. Furthermore, the extramembrane was essential for cell expansion and remodeling of the plasma membrane topology. Therefore, Myo1g is important during the recycling of lipid rafts to the membrane and to the accompanied proteins that regulate plasma membrane plasticity. Thus, Myosin 1g contributes to cell adhesion and cell migration through CD44 recycling in B lymphocytes.

  20. Functions of myosin motors tailored for parasitism

    DEFF Research Database (Denmark)

    Mueller, Christina; Graindorge, Arnault; Soldati-Favre, Dominique

    2017-01-01

    Myosin motors are one of the largest protein families in eukaryotes that exhibit divergent cellular functions. Their roles in protozoans, a diverse group of anciently diverged, single celled organisms with many prominent members known to be parasitic and to cause diseases in human and livestock......, are largely unknown. In the recent years many different approaches, among them whole genome sequencing, phylogenetic analyses and functional studies have increased our understanding on the distribution, protein architecture and function of unconventional myosin motors in protozoan parasites. In Apicomplexa......, myosins turn out to be highly specialized and to exhibit unique functions tailored to accommodate the lifestyle of these parasites....

  1. HIV infection of T cells: actin-in and actin-out.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Shaw, Stephen

    2009-04-14

    Three studies shed light on the decade-old observation that the actin cytoskeleton is hijacked to facilitate entry of HIV into its target cells. Polymerization of actin is required to assemble high concentrations of CD4 and CXCR4 at the plasma membrane, which promote viral binding and entry in both the simple model of infection by free virus and the more physiologically relevant route of infection through the virological synapse. Three types of actin-interacting proteins-filamin, ezrin/radixin/moesin (ERM), and cofilin-are now shown to play critical roles in this process. Filamin binds to both CD4 and CXCR4 in a manner promoted by signaling of the HIV gp120 glycoprotein. ERM proteins attach actin filaments to the membrane and may promote polymerization of actin. Early in the process of viral entry, cofilin is inactivated, which is proposed to facilitate the early assembly of actin filaments, but cofilin is reported to be activated soon thereafter to facilitate postentry events. This complex role of cofilin may help to reconcile the paradox that actin polymerization promotes initial binding and fusion steps but inhibits some subsequent early postentry events.

  2. Actin and Endocytosis in Budding Yeast

    Science.gov (United States)

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  3. β-Arrestin regulation of myosin light chain phosphorylation promotes AT1aR-mediated cell contraction and migration.

    Directory of Open Access Journals (Sweden)

    Elie Simard

    Full Text Available Over the last decade, it has been established that G-protein-coupled receptors (GPCRs signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC, which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1 as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR, MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM. Furthermore, by employing the β-arrestin biased ligand [Sar(1,Ile(4,Ile(8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.

  4. Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

    Science.gov (United States)

    Kumar, Nitin; Gardel, Margaret

    Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

  5. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Directory of Open Access Journals (Sweden)

    José R Sotelo

    Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

  6. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  7. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, V.; Rülicke, T.; Rathkolb, B.; Hans, W.; Bohla, A.; Eickelberg, O.; Stoeger, T.; Wolf, E.; Yildirim, A.Ö.; Gailus-Durner, V.; Fuchs, H.; de Angelis, M.H.; Hozák, Pavel

    2013-01-01

    Roč. 8, č. 4 (2013), e61406 E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020022; GA MŠk LH12143; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : nuclear myosin * myosin isoforms * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  8. Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits

    Science.gov (United States)

    2016-01-01

    Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the “stiffness site” affects filament mechanical properties. Incorporating a magnesium ion in the “polymerization site” does not seem to require any large-scale change to an actin subunit’s conformation. Binding of a magnesium ion in the “stiffness site” adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

  9. Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

    Science.gov (United States)

    Weil, D; Levy, G; Sahly, I; Levi-Acobas, F; Blanchard, S; El-Amraoui, A; Crozet, F; Philippe, H; Abitbol, M; Petit, C

    1996-04-16

    The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

  10. Rethinking Molecular Mimicry in Rheumatic Heart Disease andAutoimmune Myocarditis: Laminin, Collagen IV, CAR and B1AR as Initial Targets of Disease

    Directory of Open Access Journals (Sweden)

    Robert eRoot-Bernstein

    2014-08-01

    Full Text Available Rationale: Molecular mimicry theory (MMT suggests that epitope mimicry between pathogens and human proteins can activate autoimmune disease. Group A streptococci (GAS mimics human cardiac myosin in rheumatic heart disease (RHD and coxsackie viruses (CX mimic actin in autoimmune myocarditis (AM. But myosin and actin are immunologically inaccessible and unlikely initial targets. Extracellular cardiac proteins that mimic GAS and CX would be more likely.Objectives: To determine whether extracellular cardiac proteins such as coxsackie and adenovirus receptor (CAR, beta 1 adrenergic receptor (B1AR, CD55/DAF, laminin, and collagen IV mimic GAS, CX and/or cardiac myosin or actin. Methods: BLAST 2.0 and LALIGN searches of the UniProt protein database were employed to identify potential molecular mimics. Quantitative ELISA was used to measure antibody cross-reactivity. Measurements: Similarities were considered to be significant if a sequence contained at least 5 identical amino acids in 10. Antibodies were considered to be cross-reactive if the binding constant had a Kd less than 10-9 M. Main Results: GAS mimics laminin, CAR and myosin. CX mimics actin and collagen IV and B1AR. The similarity search results are mirrored by antibody cross-reactivities. Additionally, antibodies against laminin recognize antibodies against collagen IV; antibodies against actin recognize antibodies against myosin, and antibodies against GAS recognize antibodies against CX. Thus, there is both mimicry of extracellular proteins and antigenic complementarity between GAS-CX in RHD/AM.Conclusions: RHD/AM may be due to combined infections of GAS with CX localize at cardiomyocytes may produce a synergistic, hyperinflammatory response that cross-reacts with laminin, collagen IV, CAR and/or B1AR. Epitope drift shifts the immune response to myosin and actin after cardiomyocytes become damaged.

  11. Phosphorylation of human skeletal muscle myosin

    International Nuclear Information System (INIS)

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-01-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

  12. Physical driving force of actomyosin motility based on the hydration effect.

    Science.gov (United States)

    Suzuki, Makoto; Mogami, George; Ohsugi, Hideyuki; Watanabe, Takahiro; Matubayasi, Nobuyuki

    2017-12-01

    We propose a driving force hypothesis based on previous thermodynamics, kinetics and structural data as well as additional experiments and calculations presented here on water-related phenomena in the actomyosin systems. Although Szent-Györgyi pointed out the importance of water in muscle contraction in 1951, few studies have focused on the water science of muscle because of the difficulty of analyzing hydration properties of the muscle proteins, actin, and myosin. The thermodynamics and energetics of muscle contraction are linked to the water-mediated regulation of protein-ligand and protein-protein interactions along with structural changes in protein molecules. In this study, we assume the following two points: (1) the periodic electric field distribution along an actin filament (F-actin) is unidirectionally modified upon binding of myosin subfragment 1 (M or myosin S1) with ADP and inorganic phosphate Pi (M.ADP.Pi complex) and (2) the solvation free energy of myosin S1 depends on the external electric field strength and the solvation free energy of myosin S1 in close proximity to F-actin can become the potential force to drive myosin S1 along F-actin. The first assumption is supported by integration of experimental reports. The second assumption is supported by model calculations utilizing molecular dynamics (MD) simulation to determine solvation free energies of a small organic molecule and two small proteins. MD simulations utilize the energy representation method (ER) and the roughly proportional relationship between the solvation free energy and the solvent-accessible surface area (SASA) of the protein. The estimated driving force acting on myosin S1 is as high as several piconewtons (pN), which is consistent with the experimentally observed force. © 2017 Wiley Periodicals, Inc.

  13. Myosin heavy chain expression in rabbit masseter muscle during postnatal development

    NARCIS (Netherlands)

    Bredman, J. J.; Weijs, W. A.; Korfage, H. A.; Brugman, P.; Moorman, A. F.

    1992-01-01

    The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' alpha-MHC. During postnatal growth myosin

  14. Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

    Science.gov (United States)

    Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

    2018-02-01

    Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  15. Fast and slow myosins as markers of muscle injury.

    Science.gov (United States)

    Guerrero, M; Guiu-Comadevall, M; Cadefau, J A; Parra, J; Balius, R; Estruch, A; Rodas, G; Bedini, J L; Cussó, R

    2008-07-01

    The diagnosis of muscular lesions suffered by athletes is usually made by clinical criteria combined with imaging of the lesion (ultrasonography and/or magnetic resonance) and blood tests to detect the presence of non-specific muscle markers. This study was undertaken to evaluate injury to fast and slow-twitch fibres using specific muscle markers for these fibres. Blood samples were obtained from 51 non-sports people and 38 sportsmen with skeletal muscle injury. Western blood analysis was performed to determine fast and slow myosin and creatine kinase (CK) levels. Skeletal muscle damage was diagnosed by physical examination, ultrasonography and magnetic resonance and biochemical markers. The imaging tests were found to be excellent for detecting and confirming grade II and III lesions. However, grade I lesions were often unconfirmed by these techniques. Grade I lesions have higher levels of fast myosin than slow myosin with a very small increase in CK levels. Grade II and III lesions have high values of both fast and slow myosin. The evaluation of fast and slow myosin in the blood 48 h after the lesion occurs is a useful aid for the detection of type I lesions in particular, since fast myosin is an exclusive skeletal muscle marker. The correct diagnosis of grade I lesions can prevent progression of the injury in athletes undergoing continual training sessions and competitions, thus aiding sports physicians in their decision making.

  16. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    Directory of Open Access Journals (Sweden)

    Jayabalan M Joseph

    Full Text Available Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps. To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group. According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8 as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  17. Engineering controllable bidirectional molecular motors based on myosin

    Science.gov (United States)

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

    2012-04-01

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

  18. Cardiac muscle: a miracle of creation.

    Science.gov (United States)

    Seely, S

    1989-09-01

    The paper proposes that energy conversion in muscle is a two-step process, chemical energy being first converted into electrical energy which is then converted into mechanical work. The chemo-electrical transducers are, in effect, minute voltaic cells--more precisely calcium-magnesium cells--with the magnesium electrodes on myosin heads and the calcium electrodes on the C subunits of troponin molecules associated with actin filaments. These cells are established when, after the passage of an action potential, calcium ions are admitted to the sarcomere. In an energy-consuming process, calcium ions are bound to troponin molecules, the energy for the process being supplied by hydrolysis of adenosine triphosphate. The electro-mechanical transducer utilises the electrostatic field established between the oppositely charged electrodes of the voltaic cell. As the two are pulled towards each other, doing mechanical work, energy is supplied by the voltaic cells. In the course of this action, calcium ions go back into solution. The action ceases when, after the passage of an action potential, calcium ions are withdrawn into the sarcoplasmic reticulum.

  19. Dynamics of myosin II organization into cortical contractile networks and fibers

    Science.gov (United States)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

    2014-03-01

    The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

  20. Distribution and evolution of stable single α-helices (SAH domains in myosin motor proteins.

    Directory of Open Access Journals (Sweden)

    Dominic Simm

    Full Text Available Stable single-alpha helices (SAHs are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss.

  1. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  2. Transportation of drug–gold nanocomposites by actinomyosin motor system

    International Nuclear Information System (INIS)

    Kaur, Harsimran; Chaudhary, Archana; Kaur, Inderpreet; Singh, Kashmir; Bharadwaj, Lalit M.

    2011-01-01

    Nanotechnology is playing an important role in drug delivery to overcome limitations of conventional drug delivery systems in terms of solubility, in vivo stability, pharmacokinetics, and bio-distribution. The controlled transportation of drug into the cell and within the cell is a major challenge to be addressed. Cellular molecular motors have been exploited for their cargo carrying capacity for various applications including engineering and health care. Combination of nanotechnology and biomolecular motors can address some of the challenges in drug delivery. In the present study, transportation of drug nanocomposites has been demonstrated. Nanocomposites of 6-mercaptopurine and levodopa drugs (cancer and Parkinson’s disease, respectively) were prepared with gold nanoparticles (GNPs) by covalent attachment and these nanocomposites were attached to actin filaments. These nanocomposites were in-turn transported by actin filaments on myosin tracks. Characterization of drug nanocomposites formation was done by UV–Vis spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and confocal microscopy. GNP composites of 6-mercaptopurine and levodopa were formed by sulfide and amide bond formation, respectively. Average velocity of actin filament attached to nanocomposites was found to be 3.17 and 3.89 μm/s for levodopa and 6-mercaptopurine, respectively, as compared to actin filaments with velocity of 4.0–6.0 μm/s. Three concepts have been proposed for the study of drug transportation into the cell based on polycationic complex formation, interaction of actin with cellular myosin and Biomolecular Adaptor for Retrograde Transport (BART) technology. The aspects of this study heads toward the development of an approach to utilize molecular motors for nanoscale transportation endogenously.

  3. Transportation of drug-gold nanocomposites by actinomyosin motor system

    Science.gov (United States)

    Kaur, Harsimran; Chaudhary, Archana; Kaur, Inderpreet; Singh, Kashmir; Bharadwaj, Lalit M.

    2011-06-01

    Nanotechnology is playing an important role in drug delivery to overcome limitations of conventional drug delivery systems in terms of solubility, in vivo stability, pharmacokinetics, and bio-distribution. The controlled transportation of drug into the cell and within the cell is a major challenge to be addressed. Cellular molecular motors have been exploited for their cargo carrying capacity for various applications including engineering and health care. Combination of nanotechnology and biomolecular motors can address some of the challenges in drug delivery. In the present study, transportation of drug nanocomposites has been demonstrated. Nanocomposites of 6-mercaptopurine and levodopa drugs (cancer and Parkinson's disease, respectively) were prepared with gold nanoparticles (GNPs) by covalent attachment and these nanocomposites were attached to actin filaments. These nanocomposites were in-turn transported by actin filaments on myosin tracks. Characterization of drug nanocomposites formation was done by UV-Vis spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and confocal microscopy. GNP composites of 6-mercaptopurine and levodopa were formed by sulfide and amide bond formation, respectively. Average velocity of actin filament attached to nanocomposites was found to be 3.17 and 3.89 μm/s for levodopa and 6-mercaptopurine, respectively, as compared to actin filaments with velocity of 4.0-6.0 μm/s. Three concepts have been proposed for the study of drug transportation into the cell based on polycationic complex formation, interaction of actin with cellular myosin and Biomolecular Adaptor for Retrograde Transport (BART) technology. The aspects of this study heads toward the development of an approach to utilize molecular motors for nanoscale transportation endogenously.

  4. Transportation of drug-gold nanocomposites by actinomyosin motor system

    Energy Technology Data Exchange (ETDEWEB)

    Kaur, Harsimran, E-mail: microsimbac@gmail.com; Chaudhary, Archana; Kaur, Inderpreet [Council of Scientific and Industrial Research (CSIR), Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organization - CSIO (India); Singh, Kashmir [Panjab University, Department of Biotechnology (India); Bharadwaj, Lalit M. [Council of Scientific and Industrial Research (CSIR), Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organization - CSIO (India)

    2011-06-15

    Nanotechnology is playing an important role in drug delivery to overcome limitations of conventional drug delivery systems in terms of solubility, in vivo stability, pharmacokinetics, and bio-distribution. The controlled transportation of drug into the cell and within the cell is a major challenge to be addressed. Cellular molecular motors have been exploited for their cargo carrying capacity for various applications including engineering and health care. Combination of nanotechnology and biomolecular motors can address some of the challenges in drug delivery. In the present study, transportation of drug nanocomposites has been demonstrated. Nanocomposites of 6-mercaptopurine and levodopa drugs (cancer and Parkinson's disease, respectively) were prepared with gold nanoparticles (GNPs) by covalent attachment and these nanocomposites were attached to actin filaments. These nanocomposites were in-turn transported by actin filaments on myosin tracks. Characterization of drug nanocomposites formation was done by UV-Vis spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and confocal microscopy. GNP composites of 6-mercaptopurine and levodopa were formed by sulfide and amide bond formation, respectively. Average velocity of actin filament attached to nanocomposites was found to be 3.17 and 3.89 {mu}m/s for levodopa and 6-mercaptopurine, respectively, as compared to actin filaments with velocity of 4.0-6.0 {mu}m/s. Three concepts have been proposed for the study of drug transportation into the cell based on polycationic complex formation, interaction of actin with cellular myosin and Biomolecular Adaptor for Retrograde Transport (BART) technology. The aspects of this study heads toward the development of an approach to utilize molecular motors for nanoscale transportation endogenously.

  5. Separation of actin-dependent and actin-independent lipid rafts

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Kok, Jan Willem

    2013-01-01

    Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting

  6. Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

    Directory of Open Access Journals (Sweden)

    Juliane Bremer

    2016-11-01

    Full Text Available During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

  7. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons.

    Science.gov (United States)

    Trivedi, Niraj; Ramahi, Joseph S; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A; Solecki, David J

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. We show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. We propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  8. Myosin content of individual human muscle fibers isolated by laser capture microdissection.

    Science.gov (United States)

    Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

    2016-03-01

    Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. Copyright © 2016 the American Physiological Society.

  9. Engineering controllable bidirectional molecular motors based on myosin

    Science.gov (United States)

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

    2012-01-01

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells1, and have potential applications in molecular detection and diagnostic devices2,3. Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in living cells, and yield optimized device components for complex tasks such as molecular sorting and directed assembly3. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions4,5 and other signals6. Here we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies7–11 and guided by a structural model12 for the redirected power stroke of myosin VI, we constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our general strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should enable spatiotemporal control over a range of motor properties including processivity, stride size13, and branchpoint turning14. PMID:22343382

  10. Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function

    Science.gov (United States)

    Ambrosio, Javier R.; Valverde-Islas, Laura; Nava-Castro, Karen E.; Palacios- Arreola, M. Isabel; Ostoa-Saloma, Pedro; Reynoso-Ducoing, Olivia; Escobedo, Galileo; Ruíz-Rosado, Azucena; Dominguez-Ramírez, Lenin; Morales-Montor, Jorge

    2015-01-01

    The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells. PMID:26076446

  11. From Cycling Between Coupled Reactions to the Cross-Bridge Cycle: Mechanical Power Output as an Integral Part of Energy Metabolism

    Directory of Open Access Journals (Sweden)

    Frank Diederichs

    2012-10-01

    Full Text Available ATP delivery and its usage are achieved by cycling of respective intermediates through interconnected coupled reactions. At steady state, cycling between coupled reactions always occurs at zero resistance of the whole cycle without dissipation of free energy. The cross-bridge cycle can also be described by a system of coupled reactions: one energising reaction, which energises myosin heads by coupled ATP splitting, and one de-energising reaction, which transduces free energy from myosin heads to coupled actin movement. The whole cycle of myosin heads via cross-bridge formation and dissociation proceeds at zero resistance. Dissipation of free energy from coupled reactions occurs whenever the input potential overcomes the counteracting output potential. In addition, dissipation is produced by uncoupling. This is brought about by a load dependent shortening of the cross-bridge stroke to zero, which allows isometric force generation without mechanical power output. The occurrence of maximal efficiency is caused by uncoupling. Under coupled conditions, Hill’s equation (velocity as a function of load is fulfilled. In addition, force and shortening velocity both depend on [Ca2+]. Muscular fatigue is triggered when ATP consumption overcomes ATP delivery. As a result, the substrate of the cycle, [MgATP2−], is reduced. This leads to a switch off of cycling and ATP consumption, so that a recovery of [ATP] is possible. In this way a potentially harmful, persistent low energy state of the cell can be avoided.

  12. A novel three-filament model of force generation in eccentric contraction of skeletal muscles.

    Directory of Open Access Journals (Sweden)

    Gudrun Schappacher-Tilp

    Full Text Available We propose and examine a three filament model of skeletal muscle force generation, thereby extending classical cross-bridge models by involving titin-actin interaction upon active force production. In regions with optimal actin-myosin overlap, the model does not alter energy and force predictions of cross-bridge models for isometric contractions. However, in contrast to cross-bridge models, the three filament model accurately predicts history-dependent force generation in half sarcomeres for eccentric and concentric contractions, and predicts the activation-dependent forces for stretches beyond actin-myosin filament overlap.

  13. Myosin VIII regulates protonemal patterning and developmental timing in the moss Physcomitrella patens.

    Science.gov (United States)

    Wu, Shu-Zon; Ritchie, Julie A; Pan, Ai-Hong; Quatrano, Ralph S; Bezanilla, Magdalena

    2011-09-01

    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 Δmyo8ABCDE 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, Δmyo8ABCDE plants produce more side branches and form gametophores much earlier than wild-type plants. In the absence of nutrient media, Δmyo8ABCDE 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 Δmyo8ABCDE plants grown in the absence of nutrients. This result, together with defects in protonemal branching, smaller caulonemal cells, and accelerated development in the Δmyo8ABCDE plants, suggests that myosin VIII is involved in hormone homeostasis in P. patens.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    The UNC-45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC-45–myosin complex using various...... is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosinactin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15–16 hypertrophic cardiomyopathy...... mutations and provide directions for drug targeting. © 2013 Wiley Periodicals, Inc....

  15. Actin Cytoskeleton-Based Plant Synapse as Gravitransducer in the Transition Zone of the Root Apex

    Science.gov (United States)

    Baluska, Frantisek; Barlow, Peter; Volkmann, Dieter; Mancuso, Stefano

    The actin cytoskeleton was originally proposed to act as the signal transducer in the plant gravity sensory-motoric circuit. Surprisingly, however, several studies have documented that roots perfom gravisensing and gravitropism more effectively if exposed to diverse anti-F-actin drugs. Our study, using decapped maize root apices, has revealed that depolymerization of F-actin stimulates gravity perception in cells of the transition zone where root gravitropism is initiated (Mancuso et al. 2006). It has been proposed (Balǔka et al. 2005, 2009a) that s the non-growing adhesive end-poles, enriched with F-actin and myosin VIII, and active in endocytic recycling of both PIN transporters and cell wall pectins cross-linked with calcium and boron, act as the gravisensing domains, and that these impinge directly upon the root motoric responses via control of polar auxin transport. This model suggests that mechanical asymmetry at these plant synapses determines vectorial gravity-controlled auxin transport. Due to the gravity-imposed mechanical load upon the protoplast, a tensional stress is also imposed upon the plasma membrane of the physically lower synaptic cell pole. This stress is then relieved by shifting the endocytosis-exocytosis balance towards exocytosis (Balǔka et al. s 2005, 2009a,b). This `Synaptic Auxin Secretion' hypothesis does not conflict with the `Starch Statolith' hypothesis, which is based on amyloplast sedimentation. In fact, the `Synaptic Auxin Secretion' hypothesis has many elements which allow its unification with the Starch-Statolith model (Balǔka et al. 2005, 2009a,b). s References Balǔka F, Volkmann D, Menzel D (2005) Plant synapses: actin-based adhesion s domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 Balǔka F, Schlicht M, s Wan Y-L, Burbach C, Volkmann D (2009a) Intracellular domains and polarity in root apices: from synaptic domains to plant neurobiology. Nova Acta Leopoldina 96: 103-122 Balǔka s F, Mancuso S

  16. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

    Science.gov (United States)

    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  17. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin or polymeric form (F-actin. Members of the actin-depolymerizing factor (ADF/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1 in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

  18. A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Morgan-Fisher, Marie; Wait, Robin

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among...... nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two......-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions...

  19. Thick Filament Protein Network, Functions, and Disease Association.

    Science.gov (United States)

    Wang, Li; Geist, Janelle; Grogan, Alyssa; Hu, Li-Yen R; Kontrogianni-Konstantopoulos, Aikaterini

    2018-03-13

    Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018. Copyright © 2018 American Physiological Society. All rights reserved.

  20. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...... dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton....

  1. The expression of myosin genes in developing skeletal muscle in the mouse embryo

    International Nuclear Information System (INIS)

    Lyons, G.E.; Ontell, M.; Cox, R.; Sassoon, D.; Buckingham, M.

    1990-01-01

    Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and acculumation

  2. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    Science.gov (United States)

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

  3. Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

    Directory of Open Access Journals (Sweden)

    Alexander E Urban

    Full Text Available Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

  4. MYBPC3 mutations are associated with a reduced super-relaxed state in patients with hypertrophic cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    James W McNamara

    Full Text Available The "super-relaxed state" (SRX of myosin represents a 'reserve' of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein-C (cMyBP-C. Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3 using samples from human patients with hypertrophic cardiomyopathy (HCM. Left ventricular (LV samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut or negative (HCMsmn and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01 between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

  6. Controllable molecular motors engineered from myosin and RNA

    Science.gov (United States)

    Omabegho, Tosan; Gurel, Pinar S.; Cheng, Clarence Y.; Kim, Laura Y.; Ruijgrok, Paul V.; Das, Rhiju; Alushin, Gregory M.; Bryant, Zev

    2018-01-01

    Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3-5 and modified natural protein motors6-10 have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors11-15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s-1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

  7. Actin filaments as tension sensors.

    Science.gov (United States)

    Galkin, Vitold E; Orlova, Albina; Egelman, Edward H

    2012-02-07

    The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture

    Science.gov (United States)

    Kubis, Hans-Peter; Haller, Ernst-August; Wetzel, Petra; Gros, Gerolf

    1997-01-01

    A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. PMID:9108130

  9. Actinic keratosis

    DEFF Research Database (Denmark)

    Erlendsson, Andrés M; Egekvist, Henrik; Lorentzen, Henrik F.

    2016-01-01

    Objectives: The incidence of actinic keratosis (AK) is increasing, and several treatment options are available. The aim of this study was to describe clinical characteristics and treatment patterns in patients with AK treated by Danish dermatologists. Methods: A multicenter, non-interventional, c......Objectives: The incidence of actinic keratosis (AK) is increasing, and several treatment options are available. The aim of this study was to describe clinical characteristics and treatment patterns in patients with AK treated by Danish dermatologists. Methods: A multicenter, non...... and currently suspected in 9.4% of AK-affected anatomical regions. Lesions of AK were located primarily on the face (38.6%), scalp (12.8%), and hands (11.2%). Actinic keratosis commonly presented with multiple AK lesions (38.6%) and field cancerization (38.5%). The treatments used most frequently were...

  10. Bacterial actin MreB forms antiparallel double filaments.

    Science.gov (United States)

    van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay Am; Johnson, Christopher M; Löwe, Jan

    2014-05-02

    Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments.DOI: http://dx.doi.org/10.7554/eLife.02634.001. Copyright © 2014, van den Ent et al.

  11. The Intriguing Dual Lattices of the Myosin Filaments in Vertebrate Striated Muscles: Evolution and Advantage

    Directory of Open Access Journals (Sweden)

    Pradeep K. Luther

    2014-12-01

    Full Text Available Myosin filaments in vertebrate striated muscle have a long roughly cylindrical backbone with cross-bridge projections on the surfaces of both halves except for a short central bare zone. In the middle of this central region the filaments are cross-linked by the M-band which holds them in a well-defined hexagonal lattice in the muscle A-band. During muscular contraction the M-band-defined rotation of the myosin filaments around their long axes influences the interactions that the cross-bridges can make with the neighbouring actin filaments. We can visualise this filament rotation by electron microscopy of thin cross-sections in the bare-region immediately adjacent to the M-band where the filament profiles are distinctly triangular. In the muscles of teleost fishes, the thick filament triangular profiles have a single orientation giving what we call the simple lattice. In other vertebrates, for example all the tetrapods, the thick filaments have one of two orientations where the triangles point in opposite directions (they are rotated by 60° or 180° according to set rules. Such a distribution cannot be developed in an ordered fashion across a large 2D lattice, but there are small domains of superlattice such that the next-nearest neighbouring thick filaments often have the same orientation. We believe that this difference in the lattice forms can lead to different contractile behaviours. Here we provide a historical review, and when appropriate cite recent work related to the emergence of the simple and superlattice forms by examining the muscles of several species ranging back to primitive vertebrates and we discuss the functional differences that the two lattice forms may have.

  12. Regimes of wave type patterning driven by refractory actin feedback: transition from static polarization to dynamic wave behaviour

    International Nuclear Information System (INIS)

    Holmes, W R; Edelstein-Keshet, L; Carlsson, A E

    2012-01-01

    Patterns of waves, patches, and peaks of actin are observed experimentally in many living cells. Models of this phenomenon have been based on the interplay between filamentous actin (F-actin) and its nucleation promoting factors (NPFs) that activate the Arp2/3 complex. Here we present an alternative biologically-motivated model for F-actin-NPF interaction based on properties of GTPases acting as NPFs. GTPases (such as Cdc42, Rac) are known to promote actin nucleation, and to have active membrane-bound and inactive cytosolic forms. The model is a natural extension of a previous mathematical mini-model of small GTPases that generates static cell polarization. Like other modellers, we assume that F-actin negative feedback shapes the observed patterns by suppressing the trailing edge of NPF-generated wave-fronts, hence localizing the activity spatially. We find that our NPF-actin model generates a rich set of behaviours, spanning a transition from static polarization to single pulses, reflecting waves, wave trains, and oscillations localized at the cell edge. The model is developed with simplicity in mind to investigate the interaction between nucleation promoting factor kinetics and negative feedback. It explains distinct types of pattern initiation mechanisms, and identifies parameter regimes corresponding to distinct behaviours. We show that weak actin feedback yields static patterning, moderate feedback yields dynamical behaviour such as travelling waves, and strong feedback can lead to wave trains or total suppression of patterning. We use a recently introduced nonlinear bifurcation analysis to explore the parameter space of this model and predict its behaviour with simulations validating those results. (paper)

  13. 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.

  14. Polycation induced actin bundles

    OpenAIRE

    Muhlrad, Andras; Grintsevich, Elena E.; Reisler, Emil

    2011-01-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations an...

  15. Coordination by Cdc42 of Actin, Contractility, and Adhesion for Melanoblast Movement in Mouse Skin

    DEFF Research Database (Denmark)

    Woodham, Emma F; Paul, Nikki R; Tyrrell, Benjamin

    2017-01-01

    traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates...... multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large...... null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics....

  16. Geometrical Determinants of Neuronal Actin Waves.

    Science.gov (United States)

    Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M; Gov, Nir S; Villard, Catherine

    2017-01-01

    Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments.

  17. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  18. Analysis of positional isotope exchange in ATP by cleavage of the βP-OγP bond. Demonstration of negligible positional isotope exchange by myosin

    International Nuclear Information System (INIS)

    Dale, M.P.; Hackney, D.D.

    1987-01-01

    A method for analysis of positional isotope exchange (PIX) during ATP ↔ HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the βP-OγP bond. This cleavage yields P/sub i/ derived from the γ-phosphoryl of ATP that contains all four of the γ oxygens. Both PIX between the β, γ-bridge and β-nonbridge positions and washout of the γ-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17 O at the β-nonbridge positions and 18 O at the β,γ-bridge and γ-nonbridge positions. Application of this method to ATP ↔ HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of γ-nonbridge oxygens in the virtual absence of PIX. At 25 0 C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s -1 , which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the β-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 0 C, this fraction correspond to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. with subfragment 1

  19. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    OpenAIRE

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the ex...

  20. Actinic keratosis among seafarers.

    Science.gov (United States)

    Oldenburg, M; Kuechmeister, B; Ohnemus, U; Baur, X; Moll, I

    2013-11-01

    The aim of this study was to assess the prevalence of UV-induced actinic keratosis and further skin lesions. A newly developed questionnaire about lifetime UV radiation exposure was completed by 514 seafarers. An experienced dermatologist inspected the whole-body skin status of all participants. The questionnaire revealed a pre-employment UV radiation exposure in 104 seafarers, sunbed use in 26 subjects and a median work-related UV radiation exposure at sea of 20 years. The diagnosis of actinic keratoses was made in 94 seafarers and the clinical diagnosis of skin cancers in 48 seafarers (28 basal cell carcinoma, 11 squamous cell carcinoma, 9 malignant melanoma). After age standardisation according to a European reference population, the male European seafarers in this study had a 1.80-fold increased risk of actinic keratosis. Actinic keratoses [OR 1.03 (1.01-1.05)] and squamous cell carcinoma [OR 1.07 (1.01-1.13)] were related to the duration of seafaring time in years. A significant association was also found between actinic keratosis/squamous cell carcinoma and sunlight exposure during home leave [OR 1.67 (1.03-2.81) and OR 6.19 (1.18-32.40)]. Furthermore, the engine room personnel-especially the technical officers-were at higher risk of developing actinic keratosis. Due to the high prevalence of actinic keratosis especially among older seafarers with fair skin, with longer duration of seafaring employment at sea and with higher UV exposure during home leave, more intensive advice should be given on sun protection both at sea and ashore.

  1. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that

  2. Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Leonardo Nogara

    Full Text Available In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity.

  3. Bacterial Actins.

    Science.gov (United States)

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

    A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

  4. Dendritic Actin Cytoskeleton: Structure, Functions, and Regulations

    Directory of Open Access Journals (Sweden)

    Anja Konietzny

    2017-05-01

    Full Text Available Actin is a versatile and ubiquitous cytoskeletal protein that plays a major role in both the establishment and the maintenance of neuronal polarity. For a long time, the most prominent roles that were attributed to actin in neurons were the movement of growth cones, polarized cargo sorting at the axon initial segment, and the dynamic plasticity of dendritic spines, since those compartments contain large accumulations of actin filaments (F-actin that can be readily visualized using electron- and fluorescence microscopy. With the development of super-resolution microscopy in the past few years, previously unknown structures of the actin cytoskeleton have been uncovered: a periodic lattice consisting of actin and spectrin seems to pervade not only the whole axon, but also dendrites and even the necks of dendritic spines. Apart from that striking feature, patches of F-actin and deep actin filament bundles have been described along the lengths of neurites. So far, research has been focused on the specific roles of actin in the axon, while it is becoming more and more apparent that in the dendrite, actin is not only confined to dendritic spines, but serves many additional and important functions. In this review, we focus on recent developments regarding the role of actin in dendrite morphology, the regulation of actin dynamics by internal and external factors, and the role of F-actin in dendritic protein trafficking.

  5. Fully-coupled mathematical modeling of actomyosin-cytosolic two-phase flow in a highly deformable moving Keratocyte cell.

    Science.gov (United States)

    Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

    2018-01-23

    Interaction between intracellular dynamics and extracellular matrix (ECM) generally occurred into very thin fragment of moving cell, namely lamellipodia, enables all movable cells to crawl on ECM. In fast-moving cells such as fish Keratocytes, Lamellipodia including most cell area finds a fan-like shape during migration, with a variety of aspect ratio function of fish type. In this work, our purpose is to present a novel and more complete two-dimensional continuum mathematical model of actomyosin-cytosolic two-phase flow of a self-deforming Keratocyte with circular spreaded to steady fan-like shape. In the new approach, in addition to the two-phase flow of the F-actin and cytosol, the G-actin transport was spatiotemporally modeled. We also for the first time modeled the effect of variable volume fraction of the moving F-actin porous network on solute transport in the cytosolic fluid. Our novel fully-coupled mathematical model provides a better understanding of intracellular dynamics of fast-migrating Keratocytes; such as the F-actin centripetal and cytosolic fountain-like flows, free-active myosin distribution, distribution sequence of the G-actin, F-actin, and myosin, and myosin-induced pressure flied of cytoplasm as well as the map of intracellular forces like myosin contraction and adhesion traction. All these results are qualitatively and quantitatively in good agreement with experimental observations. According to a range of value of parameters used in this model, our steady state of moving Keratocyte finds fan-like shape with the same aspect ratio as wide category of fish Keratocytes. This new model can predict shape of Keratocytes in other range of parameter values. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Using electrical and optical tweezers to facilitate studies of molecular motors†

    Science.gov (United States)

    Arsenault, Mark E.; Sun, Yujie; Bau, Haim H.; Goldman, Yale E.

    2013-01-01

    Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells. PMID:19506758

  7. Using electrical and optical tweezers to facilitate studies of molecular motors.

    Science.gov (United States)

    Arsenault, Mark E; Sun, Yujie; Bau, Haim H; Goldman, Yale E

    2009-06-28

    Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.

  8. Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation

    DEFF Research Database (Denmark)

    Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.

    2017-01-01

    optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse...

  9. Mesoscopic model of actin-based propulsion.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.

  10. Polycation induced actin bundles.

    Science.gov (United States)

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    Science.gov (United States)

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. © 2015 Wiley Periodicals, Inc.

  12. Time-resolved x-ray diffraction studies on the intensity changes of the 5.9 and 5.1 nm actin layer lines from frog skeletal muscle during an isometric tetanus using synchrotron radiation

    International Nuclear Information System (INIS)

    Wakabayashi, K.; Tanaka, H.; Amemiya, Y.; Fujishima, A.; Kobayashi, T.; Hamanaka, T.; Sugi, H.; Mitsui, T.

    1985-01-01

    Time-resolved x-ray diffraction studies have been made on the 5.9- and 5.1-nm actin layer lines from frog skeletal muscles during an isometric tetanus at 6 degrees C, using synchrotron radiation. The integrated intensities of these actin layer lines were found to increase during a tetanus by 30-50% for the 5.9-nm reflection and approximately 70% for the 5.1-nm reflection of the resting values. The intensity increase of both reflections was greater than that taking place in the transition from rest to rigor state. The intensity change of the 5.9-nm reflection preceded those of the myosin 42.9-nm off-meridional reflection and of the equatorial reflections, as well as the isometric tension development. The intensity profile of the 5.9-nm layer line during contraction was found to be different from that observed in the rigor state

  13. Diclofenac Topical (actinic keratosis)

    Science.gov (United States)

    ... topical gel (Solaraze) is used to treat actinic keratosis (flat, scaly growths on the skin caused by ... The way diclofenac gel works to treat actinic keratosis is not known.Diclofenac is also available as ...

  14. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-01-15

    The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells. © 2017 Baird et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. Reduction in beta-myosin heavy chains in stunned myocardium as assessed by nondenaturing gel electrophoresis.

    Science.gov (United States)

    Garcia, S C; Pomblum, V J; Gams, E; Rupp, H; Schipke, J D

    2007-09-01

    Myosin plays a key role in the structure and function of cardiac muscle. Three myosin isoenzymes (V(1), V(2), and V(3)) with different ATPase activities have been identified in mammalian ventricles based on their heavy chain constituents. The relative amount of myosin isoenzymes changes under physiological and pathological conditions. Until now, myosin isoenzymes have frequently been determined using either tube gel (nondenaturing) polyacrylamide gel electrophoresis (PAGE), or gradient or uniform sodium dodecyl sulfate (denaturing) PAGE. Both methods have disadvantages, e.g., a long running time. We developed, therefore, a uniform, nondenaturing PAGE with slab minigel format for analyzing the myosin isoenzymes in normoxic and stunned rabbit hearts. In normoxic hearts of adult rabbits, V(3) predominated over V(1) (46 vs 41%). In turn, in the stunned hearts, V(1) predominated over V(3) (70 vs 30%), and the heterodimeric V(2) was not anymore detectable. This alteration appears to result from a selective loss of myosin heavy chain (MHC)-beta. In parallel, the biochemical markers troponin I and creatine kinase were increased in the stunned hearts. We suggest that alterations of myosin isoenzymes in stunned myocardium can be monitored with native PAGE. The present analysis of myosin isoenzyme appears thus as a new tool for evaluating defects in MHC dimer formation in postischemic hearts.

  17. Bifunctional Rhodamine Probes of Myosin Regulatory Light Chain Orientation in Relaxed Skeletal Muscle Fibers

    Science.gov (United States)

    Brack, Andrew S.; Brandmeier, Birgit D.; Ferguson, Roisean E.; Criddle, Susan; Dale, Robert E.; Irving, Malcolm

    2004-01-01

    The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30°C; ∼60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution—the maximum entropy distribution—consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the “lever” axis, linking Cys707 and Lys843 of the myosin heavy chain, was at 70–80° to the fiber axis, and the “hook” helix (Pro830–Lys843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region. PMID:15041671

  18. Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

    Science.gov (United States)

    Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

    2016-01-01

    The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

  19. Presence of an SH2 domain in the actin-binding protein tensin.

    Science.gov (United States)

    Davis, S; Lu, M L; Lo, S H; Lin, S; Butler, J A; Druker, B J; Roberts, T M; An, Q; Chen, L B

    1991-05-03

    The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

  20. Drosophila homologues of adenomatous polyposis coli (APC) and the formin diaphanous collaborate by a conserved mechanism to stimulate actin filament assembly.

    Science.gov (United States)

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L; McCartney, Brooke M

    2013-05-10

    Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad

  1. An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2009-01-01

    The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

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

    Science.gov (United States)

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

    2017-07-07

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

  3. Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

    Science.gov (United States)

    Nie, Wei

    The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

  4. Geometrical Determinants of Neuronal Actin Waves

    OpenAIRE

    Tomba, Caterina; Bra?ni, C?line; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M.; Gov, Nir S.; Villard, Catherine

    2017-01-01

    Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time betwe...

  5. Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

    Science.gov (United States)

    Hansen, Scott D.; Zuchero, J. Bradley; Mullins, R. Dyche

    2014-01-01

    The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy. PMID:23868587

  6. Unconventional actin conformations localize on intermediate filaments in mitosis

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. → These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). → Mitotic unconventional actin cables are independent of filamentous actin or microtubules. → Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  7. Excessive Myosin Activity in Mbs Mutants Causes Photoreceptor Movement Out of the Drosophila Eye Disc Epithelium

    OpenAIRE

    Lee, Arnold; Treisman, Jessica E.

    2004-01-01

    Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc e...

  8. Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala.

    Science.gov (United States)

    Lamprecht, R; Margulies, D S; Farb, C R; Hou, M; Johnson, L R; LeDoux, J E

    2006-01-01

    Learning and memory depend on signaling molecules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the training stimuli were presented in a non-associative manner. Anatomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically implicated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nucleus of the amygdala. When ML-7 was applied without associative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the circuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

  9. Boolean gates on actin filaments

    International Nuclear Information System (INIS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications. - Highlights: • We simulate interaction between voltage pulses using on actin filaments. • We use a coupled nonlinear transmission line model. • We design Boolean logical gates via interactions between the voltage pulses. • We construct one-bit half-adder with interacting voltage pulses.

  10. Boolean gates on actin filaments

    Energy Technology Data Exchange (ETDEWEB)

    Siccardi, Stefano, E-mail: ssiccardi@2ssas.it [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom); Tuszynski, Jack A., E-mail: jackt@ualberta.ca [Department of Oncology, University of Alberta, Edmonton, Alberta (Canada); Adamatzky, Andrew, E-mail: andrew.adamatzky@uwe.ac.uk [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom)

    2016-01-08

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications. - Highlights: • We simulate interaction between voltage pulses using on actin filaments. • We use a coupled nonlinear transmission line model. • We design Boolean logical gates via interactions between the voltage pulses. • We construct one-bit half-adder with interacting voltage pulses.

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

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

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

  12. Some techniques for studying actomyosin by electron microscopy; Quelques techniques d'etude de l'actomyosine par microscopie electronique

    Energy Technology Data Exchange (ETDEWEB)

    Herzberg, M [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires. Departement de Biologie

    1967-07-01

    A method has been developed for shadowing using a platinum-carbon alloy; it has been applied to the study of the interaction of actomyosin with its substrate ATP. The results obtained seem to favour the hypothesis of a dissociation of the actomyosin into its two components: actin and myosin. Pictures have been obtained showing particles resembling non-polymerized myosin but no rigorous proof of this exists. (author) [French] Une methode d'ombrage par un alliage de platine et carbone a ete mise au point et appliquee a l'etude de l'interaction de l'actomyosine et de son substrat l'ATP. Les resultats obtenus semblent pencher en faveur de la these d'une dissociation de l'actomyosine en ses deux constituants: l'actine et la myosine. Des images ont ete obtenues montrant des particules dont l'identite avec la myosine non polymerisee reste a demontrer plus rigoureusement. (auteur)

  13. 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.

  14. Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments.

    Science.gov (United States)

    Swartz, Darl R; Yang, Zhenyun; Sen, Asok; Tikunova, Svetlana B; Davis, Jonathan P

    2006-08-18

    Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on Tn

  15. Effects of a myosin light chain kinase inhibitor on the optics and accommodation of the avian crystalline lens.

    Science.gov (United States)

    Luck, Sara; Choh, Vivian

    2011-01-01

    While many studies investigate the cytoskeletal properties of the lens with respect to cataract development, examinations of how these molecular structures interact are few. Myosin light chain kinase (MLCK), actin, and myosin are present on the crystalline lenses of chickens. The purpose of this experiment was to determine whether contractile proteins found on the lens play a role in the optical functions of the lens at rest, and during accommodation. Eyes of 6-day old white Leghorn chicks (Gallus gallus domesticus) were enucleated, with the ciliary nerve intact. One eye was treated with the MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) and the other eye with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. The back vertex focal lengths (BVFLs) were measured before, during, and after accommodation using an optical laser scanning monitor (Scantox™). To further confirm ML-7 activity, western blotting was performed to detect whether MLCK was inhibited. Western blots confirmed that MLCK was inhibited at all three ML-7 concentrations. Ten µM ML-7 treatments led to longer BVFLs at rest (p=0.0338), while 100 µM treatments led to opposite changes, resulting in shorter BVFLs (p=0.0220). While 1 µM treatments did not lead to significant optical changes (p=0.4416), BVFLs were similar in pattern to those of the 10 µM group. ML-7 had no effects on accommodative amplitudes (p=0.7848). Inhibition of MLCK by ML-7 led to differential changes in BVFLs that presumably affected lenticular integrity. No apparent effect on accommodative amplitudes was observed.

  16. Actin expression in some Platyhelminthe species.

    Science.gov (United States)

    Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R

    1994-10-01

    Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

  17. [Myosin B ATPase activity of the intestinal smooth muscle in intestinal obstruction].

    Science.gov (United States)

    Takamatsu, H

    1983-06-01

    Intestinal smooth myosin B was prepared from muscle layers around the lesion in dogs with experimental colonic stenosis and in patients with congenital intestinal obstruction. Mg2+-ATPase activity of the myosin B was compared between the proximal dilated segment and distal segment to obstruction. Experimental colonic stenosis: In early period after surgery, proximal colons showed higher activity of myosin B ATPase than distal colons, decreasing to less than distal colon as time passed. Congenital intestinal obstruction: In three cases, whose atresia might have occurred at earlier period of gestation, proximal bowels showed less activity of myosin B ATPase than distal bowels. However, in two cases, whose atresia might have occurred at later period of gestation, and two cases with intestinal stenosis, proximal bowels indicated higher activity of myosin B ATPase than distal bowels. These data suggested that the contractibility of the proximal intestine was depending on the duration of obstruction, and it was depressed in the former patients and was accelerated in the latter patients. These results suggested that the extensive resection of dilated proximal bowel in the congenital atresia is not always necessary to obtain good postoperative intestinal dynamics at the operation of the atresial lesions which may be induced at later period of gestation. They also suggested that surgery for intestinal obstruction should be performed before the depression of intestinal contractibility to get good bowel function.

  18. Preparation and Characterization of Myosin Proteins.

    Science.gov (United States)

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  19. The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

    Science.gov (United States)

    2013-10-01

    2013 Accepted 14 February 2013 Available online 21 February 2013 Keywords: Myosin IC Isoforms Nucleolar localization signal Nucleolus Nucleus RNA...polymerase I Fibrillarinnt matter & 2013 Elsevier 1016/j.yexcr.2013.02.008 S, nucleolar localization ; No, nucleolus ; N, nucle bovine serum albumin; S...the nucleus, and the nucleolus . In the cytoplasm, myosin IC associ- ates with membranes and is involved in vesicle transport of membrane proteins [2

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

    Science.gov (United States)

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

    1992-01-01

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

  1. Actin dynamics and the elasticity of cytoskeletal networks

    Directory of Open Access Journals (Sweden)

    2009-09-01

    Full Text Available The structural integrity of a cell depends on its cytoskeleton, which includes an actin network. This network is transient and depends upon the continual polymerization and depolymerization of actin. The degradation of an actin network, and a corresponding reduction in cell stiffness, can indicate the presence of disease. Numerical simulations will be invaluable for understanding the physics of these systems and the correlation between actin dynamics and elasticity. Here we develop a model that is capable of generating actin network structures. In particular, we develop a model of actin dynamics which considers the polymerization, depolymerization, nucleation, severing, and capping of actin filaments. The structures obtained are then fed directly into a mechanical model. This allows us to qualitatively assess the effects of changing various parameters associated with actin dynamics on the elasticity of the material.

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

    International Nuclear Information System (INIS)

    Bapat, K.; Venkatesh, M.; Pillai, M.R.A.; Sarma, H.D.; Sainis, K.B.

    1998-01-01

    Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125 I and 99m Tc. 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 125 I 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 99m Tc 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 125 I and 99m Tc 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)

  3. The nature of the globular- to fibrous-actin transition.

    Science.gov (United States)

    Oda, Toshiro; Iwasa, Mitsusada; Aihara, Tomoki; Maéda, Yuichiro; Narita, Akihiro

    2009-01-22

    Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

  4. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

    Science.gov (United States)

    Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

  5. The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

    Science.gov (United States)

    Balsamo, Janne; Arregui, Carlos; Leung, TinChung; Lilien, Jack

    1998-01-01

    Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion. PMID:9786960

  6. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

    Directory of Open Access Journals (Sweden)

    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  7. Towards bridging the gap from molecular forces to the movement of organisms

    DEFF Research Database (Denmark)

    Nielsen, Bjørn Gilbert

    2004-01-01

    Muscles are responsible for generating the forces required for the movement of multicellular organisms. Microscopically, these forces arise as a consequence of motor proteins (myosin) pulling and sliding along actin filaments. Current knowledge states that the molecular forces between actin...

  8. Metal cation controls phosphate release in the myosin ATPase.

    Science.gov (United States)

    Ge, Jinghua; Huang, Furong; Nesmelov, Yuri E

    2017-11-01

    Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium "holds" phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin-nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate. © 2017 The Protein Society.

  9. K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

    International Nuclear Information System (INIS)

    Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

    1988-01-01

    Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca 2+ -dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca 2+ -independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10 -6 M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10 -4 M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 μM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [γ- 32 P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase

  10. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    International Nuclear Information System (INIS)

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J.

    1990-01-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation

  11. The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

    Science.gov (United States)

    He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

    2015-01-01

    Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

  12. Managing actinic keratosis in primary care.

    Science.gov (United States)

    Salmon, Nicola; Tidman, Michael J

    2016-10-01

    Actinic, or solar, keratosis is caused by chronic ultraviolet-induced damage to the epidermis. In the UK, 15-23% of individuals have actinic keratosis lesions. Risk factors include: advanced age; male gender; cumulative sun exposure or phototherapy; Fitzpatrick skin phototypes I-II; long-term immuno-suppression and genetic syndromes e.g. xeroderma pigmentosum and albinism. Actinic keratoses are regarded by some authorities as premalignant lesions that may transform into invasive squamous cell carcinoma (SCC) and by others as in situ SCC that may progress to an invasive stage. The risk of malignant change appears low; up to 0.5% per lesion per year. Up to 20-30% of lesions may spontaneously regress but in the absence of any reliable prognostic clinical indicators regarding malignant potential active treatment is considered appropriate. Actinic keratosis lesions may present as discrete hyperkeratotic papules, cutaneous horns, or more subtle flat lesions on sun-exposed areas of skin. The single most helpful diagnostic sign is an irregularly roughened surface texture: a sandpaper-like feel almost always indicates actinic damage. Dermatoscopy can be helpful in excluding signs of basal cell carcinoma when actinic keratosis is non-keratotic. It is always important to consider the possibility of SCC. The principal indication for referral to secondary care is the possibility of cutaneous malignancy. However, widespread and severe actinic damage in patients who are immunosuppressed is also a reason for referral.

  13. Bioinformatics study of the mangrove actin genes

    Science.gov (United States)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  14. Apatite-mediated actin dynamics in resorbing osteoclasts.

    Science.gov (United States)

    Saltel, Frédéric; Destaing, Olivier; Bard, Frédéric; Eichert, Diane; Jurdic, Pierre

    2004-12-01

    The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

  15. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    Science.gov (United States)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  16. Stretch activates myosin light chain kinase in arterial smooth muscle

    International Nuclear Information System (INIS)

    Barany, K.; Rokolya, A.; Barany, M.

    1990-01-01

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively

  17. Generation of life in a test tube: Albert Szent-Gyorgyi, Bruno Straub, and the discovery of actin.

    Science.gov (United States)

    Rall, Jack A

    2018-06-01

    This is a story about a great scientist, luck, great discovery that changed the future direction of muscle research, war, a clandestine war mission, postwar politics, and an attempt to rewrite scientific history. Albert Szent-Gyorgyi, at 44 yr of age, won the Nobel Prize in 1937 for his work on vitamin C and the establishment of the groundwork of the citric acid cycle. He now wanted to investigate one of the fundamental aspects of life and settled on the study of muscle contraction. The Szent-Gyorgyi laboratory in Hungary during World War II demonstrated that contraction could be reproduced in vitro by threads consisting of just two proteins, myosin and the newly discovered protein by Bruno Straub that they called actin. Szent-Gyorgyi called seeing the contraction of these threads, which occurred in the presence of ATP and ions, "the most thrilling moment" of his scientific life. This major discovery of the generation of "life" in a test tube was totally unknown for years by the rest of the world because of the war. When the discovery was finally communicated to the world, it was not immediately accepted by all as being relevant to the physiology of muscle contraction. Nonetheless, this discovery opened up the modern phase of muscle research. Serendipity played an important role in the great discovery, and much later politics would lead to a shocking controversy around the true discoverer of actin.

  18. Probing friction in actin-based motility

    International Nuclear Information System (INIS)

    Marcy, Yann; Joanny, Jean-Francois; Prost, Jacques; Sykes, Cecile

    2007-01-01

    Actin dynamics are responsible for cell protrusion and certain intracellular movements. The transient attachment of the actin filaments to a moving surface generates a friction force that resists the movement. We probe here the dynamics of these attachments by inducing a stick-slip behavior via micromanipulation of a growing actin comet. We show that general principles of adhesion and friction can explain our observations

  19. Actin filaments – a target for redox regulation

    Science.gov (United States)

    Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

    2016-01-01

    Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

  20. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty; Patel, Yashomati M.

    2008-01-01

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity

  1. Characterizing interaction forces between actin and proteins of the tropomodulin family reveals the presence of the N-terminal actin-binding site in leiomodin.

    Science.gov (United States)

    Arslan, Baran; Colpan, Mert; Gray, Kevin T; Abu-Lail, Nehal I; Kostyukova, Alla S

    2018-01-15

    Tropomodulin family of proteins includes several isoforms of tropomodulins (Tmod) and leiomodins (Lmod). These proteins can sequester actin monomers or nucleate actin polymerization. Although it is known that their actin-binding properties are isoform-dependent, knowledge on how they vary in strengths of interactions with G-actin is missing. While it is confirmed in many studies that Tmods have two actin-binding sites, information on number and location of actin-binding sites in Lmod2 is controversial. We used atomic force microscopy to study interactions between G-actin and proteins of the tropomodulin family. Unbinding forces between G-actin and Tmod1, Tmod2, Tmod3, or Lmod2 were quantified. Our results indicated that Tmod1 and Tmod3 had unimodal force distributions, Tmod2 had a bimodal distribution and Lmod2 had a trimodal distribution. The number of force distributions correlates with the proteins' abilities to sequester actin or to nucleate actin polymerization. We assigned specific unbinding forces to the individual actin-binding sites of Tmod2 and Lmod2 using mutations that destroy actin-binding sites of Tmod2 and truncated Lmod2. Our results confirm the existence of the N-terminal actin-binding site in Lmod2. Altogether, our data demonstrate how the differences between the number and the strength of actin-binding sites of Tmod or Lmod translate to their functional abilities. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Mathematical model of SPOC with a time dependent reactive field

    International Nuclear Information System (INIS)

    Ohtaki, Masako

    2006-01-01

    In general the muscle is in one of the two state possible states, relaxation or contraction. These contractions result from relative sliding of myosin and actin in the sarcomere, which is the contraction structure unit of skeletal muscle. The switching between two states is depending on the Ca2 + concentrations. However another state has been detected between these two states. In the third state, that is SPontaneous Oscillatory Contraction (SPOC), sarcomere repeats contraction and extension spontaneously. Muscle fibers are composed of hundreds of sarcomeres in series and one sarcomea also is composed of hundreds of myosin. In microscopic, the force generated by actin and myosin interaction occurs stochastically. SPOC, however, is macroscopically observable and there are regular oscillations. To understand SPOC mechanism, we propose a model for SPOC based on chemical reaction including mechanical process

  3. Molecular cloning and complete nucleotide sequence of a human ventricular myosin light chain 1

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, E; Shi, Q W; Floroff, M; Mickle, D A.G.; Wu, T W; Olley, P M; Jackowski, G

    1988-03-25

    Human ventricular plasmid library was constructed. The library was screened with the oligonucleotide probe (17-mer) corresponding to a conserve region of myosin light chain 1 near the carboxy terminal. Full length cDNA recombinant plasmid containing 1100 bp insert was isolated. RNA blot hybridization with this insert detected a message of approximately 1500 bp corresponding to the size of VLCl and mRNA. Complete nucleotide sequence of the coding region was determined in M13 subclones using dideoxy chain termination method. With the isolation of this clone (pCD HLVCl), the publication of the complete nucleotide sequence of HVLCl and the predicted secondary structure of this protein will aid in understanding of the biochemistry of myosin and its function in contraction, the evolution of myosin light genes and the genetic, developmental and physiological regulation of myosin genes.

  4. Plasmin enzymatic activity in the presence of actin

    Directory of Open Access Journals (Sweden)

    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 [γ- 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

  6. Mechanics model for actin-based motility.

    Science.gov (United States)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  7. Titin-Actin Interaction: PEVK-Actin-Based Viscosity in a Large Animal

    Directory of Open Access Journals (Sweden)

    Charles S. Chung

    2011-01-01

    Full Text Available Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5–1.0 than mice (N2BA:N2B ratio ~0.2. To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1–400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL ``overshoot’’ at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

  8. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    Science.gov (United States)

    Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

    2013-01-01

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

  9. Electrostatics control actin filament nucleation and elongation kinetics.

    Science.gov (United States)

    Crevenna, Alvaro H; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L; Lamb, Don C; Wedlich-Söldner, Roland

    2013-04-26

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

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

    NARCIS (Netherlands)

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

    2016-01-01

    textabstractMyosin 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

  11. Three-dimensional simulation of pseudopod-driven swimming of amoeboid cells

    Science.gov (United States)

    Campbell, Eric; Bagchi, Prosenjit

    2016-11-01

    Pseudopod-driven locomotion is common in eukaryotic cells, such as amoeba, neutrophils, and cancer cells. Pseudopods are protrusions of the cell body that grow, bifurcate, and retract. Due to the dynamic nature of pseudopods, the shape of a motile cell constantly changes. The actin-myosin protein dynamics is a likely mechanism for pseudopod growth. Existing theoretical models often focus on the acto-myosin dynamics, and not the whole cell shape dynamics. Here we present a full 3D simulation of pseudopod-driven motility by coupling a surface-bound reaction-diffusion (RD) model for the acto-myosin dynamics, a continuum model for the cell membrane deformation, and flow of the cytoplasmic and extracellular fluids. The whole cell is represented as a viscous fluid surrounded by a membrane. A finite-element method is used to solve the membrane deformation, and the RD model on the deforming membrane, while a finite-difference/spectral method is used to solve the flow fields inside and outside the cell. The fluid flow and cell deformation are coupled by the immersed-boundary method. The model predicts pseudopod growth, bifurcation, and retraction as observed for a swimming amoeba. The work provides insights on the role of membrane stiffness and cytoplasmic viscosity on amoeboid swimming. Funded by NSF CBET 1438255.

  12. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  13. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

    Science.gov (United States)

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

    2013-01-01

    Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

  14. The Bacterial Actin MamK

    Science.gov (United States)

    Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

    2013-01-01

    It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

  15. A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Steimle, Paul A.; Kent Fulcher, F.; Patel, Yashomati M.

    2005-01-01

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis

  16. Formin' actin in the nucleus.

    Science.gov (United States)

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner.

  17. Measurement and Analysis of in vitro Actin Polymerization

    Science.gov (United States)

    Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

    2014-01-01

    Summary The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data. PMID:23868594

  18. Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

    Directory of Open Access Journals (Sweden)

    David-Watine Brigitte

    2006-05-01

    Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

  19. Integrins in cell migration – the actin connection

    OpenAIRE

    Vicente-Manzanares, Miguel; Choi, Colin Kiwon; Horwitz, Alan Rick

    2008-01-01

    The connection between integrins and actin is driving the field of cell migration in new directions. Integrins and actin are coupled through a physical linkage, which provides traction for migration. Recent studies show the importance of this linkage in regulating adhesion organization and development. Actin polymerization orchestrates adhesion assembly near the leading edge of a migrating cell, and the dynamic cross-linking of actin filaments promotes adhesion maturat...

  20. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.

    Science.gov (United States)

    Burnette, Dylan T; Shao, Lin; Ott, Carolyn; Pasapera, Ana M; Fischer, Robert S; Baird, Michelle A; Der Loughian, Christelle; Delanoe-Ayari, Helene; Paszek, Matthew J; Davidson, Michael W; Betzig, Eric; Lippincott-Schwartz, Jennifer

    2014-04-14

    How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

  1. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Benjamin B. A. Raymond

    2018-02-01

    Full Text Available Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15 using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM, and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  2. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Raymond, Benjamin B A; Madhkoor, Ranya; Schleicher, Ina; Uphoff, Cord C; Turnbull, Lynne; Whitchurch, Cynthia B; Rohde, Manfred; Padula, Matthew P; Djordjevic, Steven P

    2018-01-01

    Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  3. Mathematical model of SPOC with a time dependent reactive field

    Energy Technology Data Exchange (ETDEWEB)

    Ohtaki, Masako [Department of Physics, School of Science and Engineering, Waseda University, Okubo, Shinjuku-ku, Tokyo 169-8555 (Japan)

    2006-03-21

    In general the muscle is in one of the two state possible states, relaxation or contraction. These contractions result from relative sliding of myosin and actin in the sarcomere, which is the contraction structure unit of skeletal muscle. The switching between two states is depending on the Ca2{sup +} concentrations. However another state has been detected between these two states. In the third state, that is SPontaneous Oscillatory Contraction (SPOC), sarcomere repeats contraction and extension spontaneously. Muscle fibers are composed of hundreds of sarcomeres in series and one sarcomea also is composed of hundreds of myosin. In microscopic, the force generated by actin and myosin interaction occurs stochastically. SPOC, however, is macroscopically observable and there are regular oscillations. To understand SPOC mechanism, we propose a model for SPOC based on chemical reaction including mechanical process.

  4. Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

    Science.gov (United States)

    Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

    2015-09-01

    The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia.

  5. Biphasic interactions between a cationic dendrimer and actin.

    Science.gov (United States)

    Ruenraroengsak, Pakatip; Florence, Alexander T

    2010-12-01

    Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.

  6. Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

    Science.gov (United States)

    Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

    2018-04-01

    Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

  7. Bacterial Actins? An Evolutionary Perspective

    Science.gov (United States)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  8. Can the lesions induced with actinic keratosis become an enigma for dermatologists? Results of an open perspective study

    Directory of Open Access Journals (Sweden)

    E. . Bujnauskajte

    2017-01-01

    Full Text Available Different face skin diseases (basal cell carcinoma, actinic keratosis, rosacea, solar elastosis, etc. could clinically manifest itself as erythematic patches, pimples or plagues. It is very hard to make the clinical exclusion in some cases of these diseases since their characters can partially overlap or certain lesion can mimic another one especially in the cases of skin areas affected with sun. Therefore, the histopathological analysis remains the “golden standard” of the dermatological diagnosis at skin diseases. Our study has shown that certified dermatologists detect actinic keratosis (AK of face/head skin of I/II levels very well. Verdicts of dermatologists and pathomorfologists are congruent on account of diagnosis in 90,7% cases. Diseases clinically excluded as AK revealed as malignant neoplasms (basal cell carcinoma in less than 1% of case lesions.

  9. α-Catenin localization and sarcomere self-organization on N-cadherin adhesive patterns are myocyte contractility driven.

    Directory of Open Access Journals (Sweden)

    Anant Chopra

    Full Text Available The N-cadherin (N-cad complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.

  10. Developmental expression of the alpha-skeletal actin gene

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    Vonk Freek J

    2008-06-01

    Full Text Available Abstract Background Actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. In higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. Expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. All members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins. Results We present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. We further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species (mouse, chicken, snake, salamander and fish. Our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. In addition, we identify many novel non-muscular expression domains including several in the central nervous system. Conclusion Our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their 3 dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. Nonetheless, we find here important differences in 3D structures, in gene architectures and identify novel expression domains for this structural and functional important gene.

  11. Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance.

    Science.gov (United States)

    Turney, Stephen G; Ahmed, Mostafa; Chandrasekar, Indra; Wysolmerski, Robert B; Goeckeler, Zoe M; Rioux, Robert M; Whitesides, George M; Bridgman, Paul C

    2016-02-01

    Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion-cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation. © 2016 Turney et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

    Science.gov (United States)

    Orfanos, Zacharias; Sparrow, John C

    2013-01-01

    During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

  13. Changes in Actin Organization During the Cytotoxic Process

    NARCIS (Netherlands)

    Radosevic, K.; Radosevic, Katarina; van Leeuwen, Anne Marie T.; Segers-Nolten, Gezina M.J.; Figdor, Carl; de Grooth, B.G.; Greve, Jan

    1994-01-01

    Changes in organization of F-actin during the cytotoxic process between NK and K562 cells have been observed and studied using confpcal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F-actin content and orientation of F-actin towards the target cell have been

  14. Antibodies to actin in autoimmune haemolytic anaemia

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    Ritzmann Mathias

    2010-03-01

    Full Text Available Abstract Background In autoimmune haemolytic anaemia (AIHA, autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity. Results Actin-reactive antibodies were found in the sera of 95% of all animals tested. The reactivity was species-specific, i.e. reactivity with porcine actin was significantly higher than with rabbit actin. Sera of animals previously immunised with the M. suis adhesion protein MSG1 showed reactivity with actin prior to infection with M. suis indicating that molecular mimicry is involved in the specific autoreactive mechanism. A potentially cross-reactive epitope was detected. Conclusions This is the first report of autoreactive anti-actin antibodies involved in the pathogenesis of autoimmune haemolytic anaemia.

  15. Structural Basis of Actin Filament Nucleation by Tandem W Domains

    Science.gov (United States)

    Chen, Xiaorui; Ni, Fengyun; Tian, Xia; Kondrashkina, Elena; Wang, Qinghua; Ma, Jianpeng

    2013-01-01

    SUMMARY Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization. PMID:23727244

  16. Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization.

    Science.gov (United States)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, H Daniel; Jedlicka, Sabrina S; Vavylonis, Dimitrios

    2015-01-01

    The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. © 2015 Wiley Periodicals, Inc.

  17. Assembly properties of the Bacillus subtilis actin, MreB.

    Science.gov (United States)

    Mayer, Joshua A; Amann, Kurt J

    2009-02-01

    The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

  18. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    We investigated the polyelectrolyte properties of actin filaments which are in interaction with myosin motors, basic participants in mechano-electrical transduction in the stereocilia of the inner ear. Here, we elaborated a model in which actin filaments play the role of guides or pathways for localized flow of calcium ions.

  19. Cracking up: symmetry breaking in cellular systems

    NARCIS (Netherlands)

    Paluch, E.; Gucht, van der J.; Sykes, C.

    2006-01-01

    The shape of animal cells is, to a large extent, determined by the cortical actin network that underlies the cell membrane. Because of the presence of myosin motors, the actin cortex is under tension, and local relaxation of this tension can result in cortical flows that lead to deformation and

  20. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O.

    2010-01-01

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties

  1. Chronic Actinic Dermatitis

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    Bengü Çevirgen Cemil

    2017-06-01

    Full Text Available Chronic actinic dermatitis (CAD is characterized by persistent eczema-like lesions, mainly on sun-exposed sites, induced by ultraviolet B, sometimes ultraviolet A, and occasionally visible light. CAD is a rare photodermatitis. It is often associated with contact allergens including airborne allergens such as fragrances, plant antigens and topical medications. A 62 year old farmer is applied with eczematous lesions restricted to sun-exposed areas. Clinical findings and histopathologic features were consistent with the diagnosis of chronic actinic dermatitis. The patient also had contact allergy to multiple allergens. We present this case to emphasize the significance of patch test on CAD treatment and the success of topical tacrolimus and azathioprine.

  2. Actinic Granuloma with Focal Segmental Glomerulosclerosis

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    Ruedee Phasukthaworn

    2016-02-01

    Full Text Available Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies.

  3. Ring closure in actin polymers

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, Supurna, E-mail: supurna@rri.res.in [Raman Research Institute, Bangalore 560080 (India); Chattopadhyay, Sebanti [Doon University, Dehradun 248001 (India)

    2017-03-18

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers. - Highlights: • Ring closure of biopolymers. • Worm like chain model. • Predictions for experiments.

  4. The evolution of compositionally and functionally distinct actin filaments.

    Science.gov (United States)

    Gunning, Peter W; Ghoshdastider, Umesh; Whitaker, Shane; Popp, David; Robinson, Robert C

    2015-06-01

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity. © 2015. Published by The Company of Biologists Ltd.

  5. Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis.

    Science.gov (United States)

    Dong, Bo; Deng, Wei; Jiang, Di

    2011-04-01

    Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

  6. Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica

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    Maria Manich

    2018-05-01

    Full Text Available Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, “dot-like” structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.

  7. MEDYAN: Mechanochemical Simulations of Contraction and Polarity Alignment in Actomyosin Networks.

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    Konstantin Popov

    2016-04-01

    Full Text Available Active matter systems, and in particular the cell cytoskeleton, exhibit complex mechanochemical dynamics that are still not well understood. While prior computational models of cytoskeletal dynamics have lead to many conceptual insights, an important niche still needs to be filled with a high-resolution structural modeling framework, which includes a minimally-complete set of cytoskeletal chemistries, stochastically treats reaction and diffusion processes in three spatial dimensions, accurately and efficiently describes mechanical deformations of the filamentous network under stresses generated by molecular motors, and deeply couples mechanics and chemistry at high spatial resolution. To address this need, we propose a novel reactive coarse-grained force field, as well as a publicly available software package, named the Mechanochemical Dynamics of Active Networks (MEDYAN, for simulating active network evolution and dynamics (available at www.medyan.org. This model can be used to study the non-linear, far from equilibrium processes in active matter systems, in particular, comprised of interacting semi-flexible polymers embedded in a solution with complex reaction-diffusion processes. In this work, we applied MEDYAN to investigate a contractile actomyosin network consisting of actin filaments, alpha-actinin cross-linking proteins, and non-muscle myosin IIA mini-filaments. We found that these systems undergo a switch-like transition in simulations from a random network to ordered, bundled structures when cross-linker concentration is increased above a threshold value, inducing contraction driven by myosin II mini-filaments. Our simulations also show how myosin II mini-filaments, in tandem with cross-linkers, can produce a range of actin filament polarity distributions and alignment, which is crucially dependent on the rate of actin filament turnover and the actin filament's resulting super-diffusive behavior in the actomyosin-cross-linker system

  8. Coordination of membrane and actin cytoskeleton dynamics during filopodia protrusion.

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    Changsong Yang

    2009-05-01

    Full Text Available Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.

  9. The conserved Tarp actin binding domain is important for chlamydial invasion.

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    Travis J Jewett

    2010-07-01

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

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

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    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.

  11. Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped following a Mechanical Perturbation

    Science.gov (United States)

    Wu, Shenping; Liu, Jun; Perz-Edwards, Robert J.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2012-01-01

    The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5–6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ∼40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ∼98% of strong-binding acto-myosin attachments present after a length perturbation are confined to “target zones” of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model

  12. The Stereociliary Paracrystal Is a Dynamic Cytoskeletal Scaffold In Vivo

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    Philsang Hwang

    2015-11-01

    Full Text Available Permanency of mechanosensory stereocilia may be the consequence of low protein turnover or rapid protein renewal. Here, we devise a system, using optical techniques in live zebrafish, to distinguish between these mechanisms. We demonstrate that the stereocilium’s abundant actin cross-linker fascin 2b exchanges, without bias or a phosphointermediate, orders of magnitude faster (t1/2 of 76.3 s than any other known hair bundle protein. To establish the logic of fascin 2b’s exchange, we examine whether filamentous actin is dynamic and detect substantial β-actin exchange within the stereocilium’s paracrystal (t1/2 of 4.08 hr. We propose that fascin 2b’s behavior may enable cross-linking at fast timescales of stereocilia vibration while noninstructively facilitating the slower process of actin exchange. Furthermore, tip protein myosin XVa fully exchanges in hours (t1/2 of 11.6 hr, indicating that delivery of myosin-associated cargo occurs in mature stereocilia. These findings suggest that stereocilia permanency is underpinned by vibrant protein exchange.

  13. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  14. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Dov, Nadav [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel); Korenstein, Rafi, E-mail: korens@post.tau.ac.il [Department of Physiology and Pharmacology, Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv (Israel)

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  15. Actin and Arp2/3 localize at the centrosome of interphase cells

    Energy Technology Data Exchange (ETDEWEB)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  16. Actin and Arp2/3 localize at the centrosome of interphase cells

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. → Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. → Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. → The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  17. The local expression of adult chicken heart myosins during development. I. The three days embryonic chicken heart

    NARCIS (Netherlands)

    Sanders, E.; Moorman, A. F.; Los, J. A.

    1984-01-01

    Immunofluorescence studies were performed on serial sections of three days embryonic chicken hearts using antibodies specific for adult atrial and ventricular myosin heavy chains respectively. The anti-ventricular myosin serum reacted with the entire myocardium showing a decreasing intensity going

  18. Incorporation of β-actin loading control into zymography.

    Science.gov (United States)

    Govindasamy, Natasha; Yan, MengJie; Jurasz, Paul

    2016-11-01

    Gelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is a 100-1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay. The protocols incorporate the loading of molecular weight markers to demarcate MMP-2/-9 from the β-actin. The first protocol utilizes the overlay of a 10% zymography gel over a 5% Tris-Glycine separating gel from which the β-actin is transferred. The second protocol involves the direct transfer of the β-actin from a single 10% zymography gel.

  19. Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

    Science.gov (United States)

    Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

    2014-01-01

    Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  20. Bacterial subversion of host actin dynamics at the plasma membrane.

    Science.gov (United States)

    Carabeo, Rey

    2011-10-01

    Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion. © 2011 Blackwell Publishing Ltd.

  1. A nucleator arms race: cellular control of actin assembly.

    Science.gov (United States)

    Campellone, Kenneth G; Welch, Matthew D

    2010-04-01

    For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

  2. Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

    International Nuclear Information System (INIS)

    Saito, Shin-ya; Feng Jue; Kira, Atsushi; Kobayashi, Jun'ichi; Ohizumi, Yasushi

    2004-01-01

    The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function

  3. Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

    Science.gov (United States)

    Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

    2010-01-01

    The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

  4. The cell wall of Arabidopsis thaliana influences actin network dynamics.

    Science.gov (United States)

    Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

    2017-07-20

    In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Surfing pathogens and the lessons learned for actin polymerization.

    Science.gov (United States)

    Frischknecht, F; Way, M

    2001-01-01

    A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia

  6. Clinical study on the time courses of serum myosin light chain I levels in patients with acute myocardial infarction

    International Nuclear Information System (INIS)

    Nakanishi, Masako; Saiki, Yasuhiko; Ui, Kazuyo

    1992-01-01

    Changes of serum myosin light chain I (Myosin LCI) concentrations and creatine kinase (CK) activities were serially measured in 23 patients with acute myocardial infarction. Intracoronary thrombolysis was performed in 14 patients (ICT group) while the remaining 9 patients were treated in the conventional manner (non ICT group). The relationships between the maximum levels of serum Myosin LCI or CK and a myocardial infarct size index or left ventricular function were evaluated in 18 patients. The myocardial infarct size index was determined by 201 Tl myocardial scintigrams performed in the chronic phase. Multiple peaks of Myosin LCI were observed in 64% (9/14) of the ICT group and the first peak in 6 of these patients appeared much earlier in the same time as CK peak than in the non-ICT group, while multiple peaks were seen only in one case in the non-ICT group. The infarct size index by 201 Tl myocardial SPECT correlated with maximum Myosin LCI levels (r=0.88, p<0.001, n=10) and CK activities (r=0.67, p<0.05, n=10). These results indicate that the measurement of serum Myosin LCI is very useful for estimating the extent of myocardial damage and suggest that myocardial degeneration occurs at a very early phase of myocardial infarction. (author)

  7. WICH, a member of WASP-interacting protein family, cross-links actin filaments

    International Nuclear Information System (INIS)

    Kato, Masayoshi; Takenawa, Tadaomi

    2005-01-01

    In yeast, Verprolin plays an important role in rearrangement of the actin cytoskeleton. There are three mammalian homologues of Verprolin, WIP, CR16, and WICH, and all of them bind actin and Wiskott-Aldrich syndrome protein (WASP) and/or neural-WASP. Here, we describe a novel function of WICH. In vitro co-sedimentation analysis revealed that WICH not only binds to actin filaments but also cross-links them. Fluorescence and electron microscopy detected that this cross-linking results in straight bundled actin filaments. Overexpression of WICH alone in cultured fibroblast caused the formation of thick actin fibers. This ability of WICH depended on its own actin cross-linking activity. Importantly, the actin cross-linking activity of WICH was modified through a direct association with N-WASP. Taken together, these data suggest that WICH induces a bundled form of actin filament with actin cross-linking activity and the association with N-WASP suppresses that activity. WICH thus appears to be a novel actin bundling protein

  8. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

  9. Automated detection of actinic keratoses in clinical photographs.

    Science.gov (United States)

    Hames, Samuel C; Sinnya, Sudipta; Tan, Jean-Marie; Morze, Conrad; Sahebian, Azadeh; Soyer, H Peter; Prow, Tarl W

    2015-01-01

    Clinical diagnosis of actinic keratosis is known to have intra- and inter-observer variability, and there is currently no non-invasive and objective measure to diagnose these lesions. The aim of this pilot study was to determine if automatically detecting and circumscribing actinic keratoses in clinical photographs is feasible. Photographs of the face and dorsal forearms were acquired in 20 volunteers from two groups: the first with at least on actinic keratosis present on the face and each arm, the second with no actinic keratoses. The photographs were automatically analysed using colour space transforms and morphological features to detect erythema. The automated output was compared with a senior consultant dermatologist's assessment of the photographs, including the intra-observer variability. Performance was assessed by the correlation between total lesions detected by automated method and dermatologist, and whether the individual lesions detected were in the same location as the dermatologist identified lesions. Additionally, the ability to limit false positives was assessed by automatic assessment of the photographs from the no actinic keratosis group in comparison to the high actinic keratosis group. The correlation between the automatic and dermatologist counts was 0.62 on the face and 0.51 on the arms, compared to the dermatologist's intra-observer variation of 0.83 and 0.93 for the same. Sensitivity of automatic detection was 39.5% on the face, 53.1% on the arms. Positive predictive values were 13.9% on the face and 39.8% on the arms. Significantly more lesions (p<0.0001) were detected in the high actinic keratosis group compared to the no actinic keratosis group. The proposed method was inferior to assessment by the dermatologist in terms of sensitivity and positive predictive value. However, this pilot study used only a single simple feature and was still able to achieve sensitivity of detection of 53.1% on the arms.This suggests that image analysis is

  10. How capping protein enhances actin filament growth and nucleation on biomimetic beads.

    Science.gov (United States)

    Wang, Ruizhe; Carlsson, Anders E

    2015-11-25

    Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

  11. Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions.

    Science.gov (United States)

    Bean, Greg J; Amann, Kurt J

    2008-01-15

    MreB is a bacterial orthologue of actin that affects cell shape, polarity, and chromosome segregation. Although a significant body of work has explored its cellular functions, we know very little about the biochemical behavior of MreB. We have cloned, overexpressed in Escherichia coli, and purified untagged MreB1 from Thermotoga maritima. We have characterized the conditions that regulate its monomer-to-polymer assembly reaction, the critical concentrations of that reaction, the manner in which MreB uses nucleotides, its stability, and the structure of the assembled polymer. MreB requires a bound purine nucleotide for polymerization and rapidly hydrolyzes it following assembly. MreB assembly contains two distinct components, one that does not require divalent cations and one that does, which may comprise the nucleation and elongation phases of assembly, respectively. MreB assembly is strongly favored by increasing temperature or protein concentration but inhibited differentially by high concentrations of monovalent salts. The polymerization rate increases and the bulk critical concentration decreases with increasing temperature, but in contrast to previous reports, MreB is capable of polymerizing across a broad range of temperatures. MreB polymers are shorter and stiffer and scatter more light than eukaryotic actin filaments. Due to rapid ATP hydrolysis and phosphate release, we suggest that most assembled MreB in cells is in the ADP-bound state. Because of only moderate differences between the ATP and ADP critical concentrations, treadmilling may occur, but we do not predict dynamic instability in cells. Because of the relatively low cellular concentration of MreB and the observed structural properties of the polymer, a single MreB assembly may exist in cells.

  12. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States); Bernstein, Sanford I., E-mail: sbernst@sciences.sdsu.edu [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States)

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  13. Toward the Structure of Dynamic Membrane-Anchored Actin Networks

    Science.gov (United States)

    Weber, Igor

    2007-01-01

    In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

  14. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

    Science.gov (United States)

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-12-30

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin*

    Science.gov (United States)

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-01-01

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. PMID:27879315

  16. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    Science.gov (United States)

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

  17. Blebbistatin, a myosin II inhibitor, suppresses Ca(2+)-induced and "sensitized"-contraction of skinned tracheal muscles from guinea pig.

    Science.gov (United States)

    Yumoto, Masatoshi; Watanabe, Masaru

    2013-01-01

    Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

  18. 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...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  19. Prevalência de lesões cutâneas actínicas em pacientes com carcinoma basocelular do segmento cefálico: um estudo caso-controle Prevalence of actinic skin lesions in patients with basal cell carcinoma of the head: a case-control study

    Directory of Open Access Journals (Sweden)

    Valquíria Pessoa Chinem

    2012-04-01

    Full Text Available OBJETIVO: Avaliar a preval��ncia de lesões cutâneas actínicas em portadores de carcinoma basocelular do segmento cefálico. MÉTODOS: Foi conduzido estudo tipo caso-controle. Os casos, constituídos por pacientes com carcinoma basocelular sólido, primário, menor que dois centímetros, no segmento cefálico; e controles, por pacientes com outras dermatoses. Foram analisadas variáveis constitucionais, comportamentais e lesões actínicas. RESULTADOS: Avaliaram-se 120 casos e 360 controles. Mílio facial (OR = 2,3, leucodermia puntacta de membros superiores (OR = 2,9 e cutis romboidalis nuchae (OR = 1,8 associaram-se à neoplasia independentemente das demais variáveis, sugerindo um fenótipo de risco. Houve ainda associação com fenótipos claros, genética familiar e exposição solar cumulativa. Queimadura solar, tabagismo e alcoolismo não foram identificados como fatores de risco. O uso de fotoprotetores não evidenciou proteção; porém, o grupo controle era composto por pacientes dermatológicos, aos quais são indicados fotoprotetores regularmente. CONCLUSÃO: Lesões actínicas foram mais prevalentes em portadores de carcinoma basocelular sólido do segmento cefálico que em controles, especialmente mílio, cutis romboidalis nuchae e leucodermia puntacta, independentemente dos demais fatores de risco conhecidos.OBJECTIVE: To evaluate the prevalence of actinic skin lesions in patients with basal cell carcinoma of the head. METHODS: A case-control study was carried out. Cases were patients with primary, solid basal cell carcinoma of the head, less than two centimeters in diameter; and as controls, patients with other dermatoses. Constitutional and behavioral variables were analyzed, as well as actinic lesions. RESULTS: One hundred twenty cases and 360 controls were evaluated. Facial milia (OR = 2.3, leukoderma punctata of the upper limbs (OR = 2.9, and cutis rhomboidalis nuchae (OR = 1.8 were associated with neoplasms regardless

  20. Navigating the plant cell: intracellular transport logistics in the green kingdom.

    Science.gov (United States)

    Geitmann, Anja; Nebenführ, Andreas

    2015-10-01

    Intracellular transport in plant cells occurs on microtubular and actin arrays. Cytoplasmic streaming, the rapid motion of plant cell organelles, is mostly driven by an actin-myosin mechanism, whereas specialized functions, such as the transport of large cargo or the assembly of a new cell wall during cell division, are performed by the microtubules. Different modes of transport are used, fast and slow, to either haul cargo over long distances or ascertain high-precision targeting, respectively. Various forms of the actin-specific motor protein myosin XI exist in plant cells and might be involved in different cellular functions. © 2015 Geitmann and Nebenführ. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. Activation of the skeletal alpha-actin promoter during muscle regeneration.

    Science.gov (United States)

    Marsh, D R; Carson, J A; Stewart, L N; Booth, F W

    1998-11-01

    Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

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

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    Nuno M. Coelho

    2017-02-01

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

  3. The effect of pyrene labelling on the thermal stability of actin filaments

    International Nuclear Information System (INIS)

    Halasi, Szulamit; Papp, Gabor; Bugyi, Beata; Barko, Szilvia; Orban, Jozsef; Ujfalusi, Zoltan; Visegrady, Balazs

    2006-01-01

    The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of ∼20 during polymerisation. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics. In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (T m ) of actin filaments from 66 to 68 deg. C. A similar effect was detected in the presence of equimolar concentration of phalloidin where the T m shifted from 79 to 82 deg. C. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions

  4. The upper bound of radiation energy in the Myers-Perry black hole collision

    International Nuclear Information System (INIS)

    Gwak, Bogeun; Lee, Bum-Hoon

    2016-01-01

    We have investigated the upper bound of the radiation energy in the head-on collision of two Myers-Perry black holes. Initially, the two black holes are far away from each other, and they become one black hole after the collision. We have obtained the upper bound of the radiation energy thermodynamically allowed in the process. The upper bound of the radiation energy is obtained in general dimensions. The radiation bound depends on the alignments of rotating axes for a given initial condition due to spin-spin interaction. We have found that the collision may not be occurred for a initially ultra-spinning black hole.

  5. The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.

    Science.gov (United States)

    Zigman, S; Rafferty, N S; Scholz, D L; Lowe, K

    1992-08-01

    The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

    Science.gov (United States)

    Carew, Josephine A.; Goyal, Raj K.; Sullivan, Maryrose P.

    2014-01-01

    The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. PMID:24516539

  7. Chitin Synthases with a Myosin Motor-Like Domain Control the Resistance of Aspergillus fumigatus to Echinocandins

    Science.gov (United States)

    Jiménez-Ortigosa, Cristina; Aimanianda, Vishukumar; Muszkieta, Laetitia; Mouyna, Isabelle; Alsteens, David; Pire, Stéphane; Beau, Remi; Krappmann, Sven; Beauvais, Anne; Dufrêne, Yves F.

    2012-01-01

    Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis. PMID:22964252

  8. Actinic Keratosis Pathogenesis Update and New Patents.

    Science.gov (United States)

    Cantisani, Carmen; Paolino, Giovanni; Melis, Marcello; Faina, Valentina; Romaniello, Federico; Didona, Dario; Cardone, Michele; Calvieri, Stefano

    2016-01-01

    Actinic keratosis is a common premalignant skin lesion. Because of its increasing incidence, several efforts have been made to earlier detectection and to improve knowledge on photocarcinogenic pathways of keratinocytes. As a consequence, recently new discoveries have been done in this field. Starting from our previous review on actinic keratosis, we reviewed the literature focusing on pathogenesis and new patents in order to highlight the most recent progresses in diagnosis and therapeutic approach. Although several efforts have been done in the field of photodamaged skin, new upgrades in diagnosis and therapy are needed to detect superficial actinic keratosis earlier, to improve the disease free survival of patient and to better treat the field cancerization.

  9. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    Science.gov (United States)

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells.

    Directory of Open Access Journals (Sweden)

    María José Olivares

    Full Text Available The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

  11. Electro-optic deflectors deliver advantages over acousto-optical deflectors in a high resolution, ultra-fast force-clamp optical trap.

    Science.gov (United States)

    Woody, Michael S; Capitanio, Marco; Ostap, E Michael; Goldman, Yale E

    2018-04-30

    We characterized experimental artifacts arising from the non-linear response of acousto-optical deflectors (AODs) in an ultra-fast force-clamp optical trap and have shown that using electro-optical deflectors (EODs) instead eliminates these artifacts. We give an example of the effects of these artifacts in our ultra-fast force clamp studies of the interaction of myosin with actin filaments. The experimental setup, based on the concept of Capitanio et al. [Nat. Methods 9, 1013-1019 (2012)] utilizes a bead-actin-bead dumbbell held in two force-clamped optical traps which apply a load to the dumbbell to move it at a constant velocity. When myosin binds to actin, the filament motion stops quickly as the total force from the optical traps is transferred to the actomyosin attachment. We found that in our setup, AODs were unsuitable for beam steering due to non-linear variations in beam intensity and deflection angle as a function of driving frequency, likely caused by low-amplitude standing acoustic waves in the deflectors. These aberrations caused instability in the force feedback loops leading to artifactual jumps in the trap position. We demonstrate that beam steering with EODs improves the performance of our instrument. Combining the superior beam-steering capability of the EODs, force acquisition via back-focal-plane interferometry, and dual high-speed FPGA-based feedback loops, we apply precise and constant loads to study the dynamics of interactions between actin and myosin. The same concept applies to studies of other biomolecular interactions.

  12. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin.

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2017-01-01

    Full Text Available Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

  13. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  14. Alterations in rat cardiac myosin isozymes induced by whole-body irradiation are prevented by 3,5,3'-L-triiodothyronine

    International Nuclear Information System (INIS)

    Litten, R.Z.; Fein, H.G.; Gainey, G.T.; Walden, T.L.; Smallridge, R.C.

    1990-01-01

    Changes in cardiac myosin isozymes and serum thyroid hormone levels were investigated in rats following 10 Gy whole-body gamma irradiation. The percent beta-myosin heavy chain increased from 21.3 ± 1.8 to 28.1 ± 6.8 (NS) at 3-day postirradiation, 37.7 ± 1.9 (P less than .001) at 6-day postirradiation, and 43.8 ± 3.3 (P less than .001) at 9-day postirradiation. Along with the change in myosin isozymes was a significant 53% decrease (P less than .001) in the serum thyroxine (T4) level by day 3 postirradiation, remaining depressed through day 9 postirradiation. The serum 3,5,3'-triiodothyronine (T3) level, however, was normal until day 9, when significant depression was also observed. In contrast, the thyroid-stimulating hormone (TSH) level was significantly increased by fourfold at day 3, returning to near normal values by day 9 postirradiation. Daily injections of physiological doses of T3 (0.3 microgram/100 g body weight) prevented the change in the myosin isozymes following whole-body irradiation. Daily pharmacological injections of T3 (3.0 micrograms/100 g body weight) to the irradiated rats produced a further decrease in the percent beta-myosin heavy chain (below control values) indicating tissue hyperthyroidism. Thus, this study suggests that the change in myosin isozymes following whole-body irradiation is caused by an alteration in thyroid hormone activity

  15. F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Petersen, J; Nielsen, O; Egel, R

    1998-01-01

    Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones. We describe the distribution of F-actin during sexual differentiation. Cortical F-actin dots have previously been shown to be restricted...... to one end of the rod shaped cell during the G1 phase of the cell cycle. Within half an hour of nitrogen starvation the distribution of cortical F-actin dots switched from being monopolar to bipolar. This was then reversed as the F-actin cytoskeleton repolarized so that cortical F-actin dots accumulated...

  16. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

    Directory of Open Access Journals (Sweden)

    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  17. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.

    Science.gov (United States)

    Leite, Sérgio Carvalho; Sampaio, Paula; Sousa, Vera Filipe; Nogueira-Rodrigues, Joana; Pinto-Costa, Rita; Peters, Luanne Laurel; Brites, Pedro; Sousa, Mónica Mendes

    2016-04-19

    The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Modelling phagosomal lipid networks that regulate actin assembly

    Directory of Open Access Journals (Sweden)

    Schwarz Roland

    2008-12-01

    Full Text Available Abstract Background When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion. Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel. Results We compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and we investigated experimentally lipid interconversion and ATP metabolism in phagosomes by using suitable radioactive compounds. In a first step, a complex lipid network interconnected by chemical reactions catalyzed by known enzymes was modelled in COPASI (Complex Pathway Simulator. However, several lines of experimental evidence indicated that only the phosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters in the model. The results also indicated that a lipid network-independent ATP-consuming activity should be included in the model. When this activity was introduced, the set of differential equations satisfactorily reproduced the experimental data. On the other hand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We therefore adopted a phenomenological (black-box approach to represent the empirical observations. We proposed that lipids and ATP influence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all the experimental data were satisfactorily fitted with a single positive parameter per lipid and ATP. Conclusion By establishing an active 'dialogue' between an

  19. Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Anna eBergs

    2016-05-01

    Full Text Available Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although specific marker proteins to visualize actin cables have been developed in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here we visualized actin cables using tropomyosin (TpmA and Lifeact fused to fluorescent proteins in Aspergillus nidulans and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.

  20. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yi Jinsoo; Schmidt, Jacob; Chien Aichi; Montemagno, Carlo D [Department of Bioengineering, University of California Los Angeles, 420 Westwood Plaza, 7523 Boelter Hall, Los Angeles, CA 90095-1600 (United States)], E-mail: montemcd@ucmail.uc.edu

    2009-02-25

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  1. Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle

    OpenAIRE

    Vlahovich, Nicole; Kee, Anthony J.; Van der Poel, Chris; Kettle, Emma; Hernandez-Deviez, Delia; Lucas, Christine; Lynch, Gordon S.; Parton, Robert G.; Gunning, Peter W.; Hardeman, Edna C.

    2009-01-01

    The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5N...

  2. A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

    OpenAIRE

    Ghanbari, H A; McCarl, R L

    1980-01-01

    A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or...

  3. n-3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation.

    Science.gov (United States)

    Hou, Tim Y; Monk, Jennifer M; Fan, Yang-Yi; Barhoumi, Rola; Chen, Yong Q; Rivera, Gonzalo M; McMurray, David N; Chapkin, Robert S

    2012-04-01

    n-3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott-Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n-3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.

  4. Topical Imiquimod in the Treatment of Conjunctival Actinic Keratosis.

    Science.gov (United States)

    Rowlands, Megan A; Giacometti, Joseph N; Servat, Javier; Materin, Miguel A; Levin, Flora

    Conjunctival actinic keratosis is rare and difficult to treat, as recurrences are common. Imiquimod, an immune response modulator, is currently Food and Drug Administration-approved for cutaneous actinic keratosis and superficial basal cell carcinomas. Emerging reports have shown it to be effective in treating some periocular and conjunctival lesions. The authors present a case of a 68-year-old white man with recurrent actinic keratosis involving the pretarsal conjunctiva, which was successfully treated with 5% topical imiquimod following previous failure with cryotherapy and interferon α-2b. The patient had ocular irritation that resolved on cessation of treatment. To the authors' knowledge, this is the first report of conjunctival actinic keratosis being treated with and successfully eradicated by topical imiquimod.

  5. Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

    Science.gov (United States)

    Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

    2013-01-01

    The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176

  6. Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

    Science.gov (United States)

    Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

    2014-10-13

    Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

  7. Resolving the role of actoymyosin contractility in cell microrheology.

    Directory of Open Access Journals (Sweden)

    Christopher M Hale

    2009-09-01

    Full Text Available Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells-stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM and particle-tracking microrheology measurements of living cells.

  8. Rab7b at the intersection of intracellular trafficking and cell migration.

    Science.gov (United States)

    Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

    2015-01-01

    Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting