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Sample records for myosin ii motor

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Measurements of Myosin-II Motor Activity During Cytokinesis in Fission Yeast.

    PubMed

    Tang, Qing; Pollard, Luther W; Lord, Matthew

    2016-01-01

    Fission yeast myosin-II (Myo2p) represents the critical actin-based motor protein that drives actomyosin ring assembly and constriction during cytokinesis. We detail three different methods to measure Myo2p motor function. Actin-activated ATPases provide a readout of actomyosin ATPase motor activity in a bulk assay; actin filament motility assays reveal the speed and efficiency of myosin-driven actin filament gliding (when motors are anchored); myosin-bead motility assays reveal the speed and efficiency of myosin ensembles traveling along actin filaments (when actin is anchored). Collectively, these methods allow us to combine the standard in vivo approaches common to fission yeast with in vitro biochemical methods to learn more about the mechanistic action of myosin-II during cytokinesis.

  4. Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    PubMed Central

    Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

    2011-01-01

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. PMID:22022566

  5. Myosin II motor activity in the lateral amygdala is required for fear memory consolidation.

    PubMed

    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 recently shown that these molecular machines mobilize F-actin in response to synaptic stimulation and learning in the hippocampus. In this study, we report that Myosin II motors in the rat lateral amygdala (LA) are essential for fear memory formation. Pretraining infusions of the Myosin II inhibitor, blebbistatin (blebb), disrupted long term memory, while short term memory was unaffected. Interestingly, both post-training and pretesting infusions had no effect on memory formation, indicating that Myosin II motors operate during or shortly after learning to promote memory consolidation. These data support the idea that Myosin II motor-force generation is a general mechanism that supports memory consolidation in the mammalian CNS.

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

    ERIC Educational Resources Information Center

    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…

  7. UCS protein Rng3p is essential for myosin-II motor activity during cytokinesis in fission yeast.

    PubMed

    Stark, Benjamin C; James, Michael L; Pollard, Luther W; Sirotkin, Vladimir; Lord, Matthew

    2013-01-01

    UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.

  8. Myosin V motor proteins

    PubMed Central

    Vale, Ronald D.

    2003-01-01

    Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how “dynamic” single molecule measurements can synergize with “static” protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine. PMID:14610051

  9. Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

    SciTech Connect

    Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen; Kerekes, Ryan A; Gleason, Shaun Scott; Hatten, Mary E

    2009-01-01

    Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.

  10. Myosin VI: a multifunctional motor.

    PubMed

    Lister, I; Roberts, R; Schmitz, S; Walker, M; Trinick, J; Veigel, C; Buss, F; Kendrick-Jones, J

    2004-11-01

    Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed. PMID:15493988

  11. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  12. The structural coupling between ATPase activation and recovery stroke in the myosin II motor

    SciTech Connect

    Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-07-01

    Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

  13. Myosin II Dynamics during Embryo Morphogenesis

    NASA Astrophysics Data System (ADS)

    Kasza, Karen

    2013-03-01

    During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

  14. Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

    PubMed Central

    Shu, Shi; Liu, Xiong; Korn, Edward D.

    2005-01-01

    Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II. PMID:15671182

  15. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  16. Myosin II Activity Softens Cells in Suspension

    PubMed Central

    Chan, Chii J.; Ekpenyong, Andrew E.; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J.; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-01-01

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  17. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

  18. Maximum limit to the number of myosin II motors participating in processive sliding of actin.

    PubMed

    Rastogi, Khushboo; Puliyakodan, Mohammed Shabeel; Pandey, Vikas; Nath, Sunil; Elangovan, Ravikrishnan

    2016-01-01

    In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads. PMID:27554800

  19. Maximum limit to the number of myosin II motors participating in processive sliding of actin

    PubMed Central

    Rastogi, Khushboo; Puliyakodan, Mohammed Shabeel; Pandey, Vikas; Nath, Sunil; Elangovan, Ravikrishnan

    2016-01-01

    In this work, we analysed processive sliding and breakage of actin filaments at various heavy meromyosin (HMM) densities and ATP concentrations in IVMA. We observed that with addition of ATP solution, the actin filaments fragmented stochastically; we then determined mean length and velocity of surviving actin filaments post breakage. Average filament length decreased with increase in HMM density at constant ATP, and increased with increase in ATP concentration at constant HMM density. Using density of HMM molecules and length of actin, we estimated the number of HMM molecules per actin filament (N) that participate in processive sliding of actin. N is solely a function of ATP concentration: 88 ± 24 and 54 ± 22 HMM molecules (mean ± S.D.) at 2 mM and 0.1 mM ATP respectively. Processive sliding of actin filament was observed only when N lay within a minimum lower limit (Nmin) and a maximum upper limit (Nmax) to the number of HMM molecules. When N < Nmin the actin filament diffused away from the surface and processivity was lost and when N > Nmax the filament underwent breakage eventually and could not sustain processive sliding. We postulate this maximum upper limit arises due to increased number of strongly bound myosin heads. PMID:27554800

  20. Myosin VI: cellular functions and motor properties.

    PubMed Central

    Roberts, Rhys; Lister, Ida; Schmitz, Stephan; Walker, Matthew; Veigel, Claudia; Trinick, John; Buss, Folma; Kendrick-Jones, John

    2004-01-01

    Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed. PMID:15647169

  1. Myosin VI: cellular functions and motor properties.

    PubMed

    Roberts, Rhys; Lister, Ida; Schmitz, Stephan; Walker, Matthew; Veigel, Claudia; Trinick, John; Buss, Folma; Kendrick-Jones, John

    2004-12-29

    Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed. PMID:15647169

  2. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

    DeGiorgis, Joseph A.; Reese, Thomas S.; Bearer, Elaine L.

    2002-01-01

    Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor. PMID:11907281

  3. Regulation of myosin II during cytokinesis in higher eukaryotes.

    PubMed

    Matsumura, Fumio

    2005-07-01

    Cellular myosin II is the principal motor responsible for cytokinesis. In higher eukaryotes, phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Because signals transmitted by the mitotic spindle coordinate key spatial and temporal aspects of cytokinesis, such signals should ultimately function to activate myosin II. Thus, it follows that identification of regulatory factors involved in MLC phosphorylation should elucidate the nature of spindle-derived regulatory signals and lead to a model for how they control cytokinesis. However, the identity of these upstream molecules remains elusive. This review (which is part of the Cytokinesis series) summarizes current views of the regulatory pathway controlling MLC phosphorylation and features four candidate molecules that are likely immediate upstream myosin regulators. I discuss proposed functions for MLCK, ROCK, citron kinase and myosin phosphatase during cytokinesis and consider the possibility of a link between these molecules and the signals transmitted by the mitotic spindle.

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

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

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

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

    PubMed Central

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-01-01

    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 HPγO42− 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. PMID:25006262

  6. Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis.

    PubMed

    Yumura, Shigehiko; Ueda, Masahiro; Sako, Yasushi; Kitanishi-Yumura, Toshiko; Yanagida, Toshio

    2008-12-01

    Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.

  7. Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin.

    PubMed

    Ito, Kohji; Yamaguchi, Yukie; Yanase, Kenji; Ichikawa, Yousuke; Yamamoto, Keiichi

    2009-12-22

    Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.

  8. Kinetic mechanism of the fastest motor protein, Chara myosin.

    PubMed

    Ito, Kohji; Ikebe, Mitsuo; Kashiyama, Taku; Mogami, Toshifumi; Kon, Takahide; Yamamoto, Keiichi

    2007-07-01

    Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin.motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s(-1), and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s(-1). From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V(max) value of the actin-activated ATPase activity of 390 s(-1). The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.

  9. How actin initiates the motor activity of Myosin.

    PubMed

    Llinas, Paola; Isabet, Tatiana; Song, Lin; Ropars, Virginie; Zong, Bin; Benisty, Hannah; Sirigu, Serena; Morris, Carl; Kikuti, Carlos; Safer, Dan; Sweeney, H Lee; Houdusse, Anne

    2015-05-26

    Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

  10. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  11. The non-linear elasticity of the muscle sarcomere and the compliance of myosin motors

    PubMed Central

    Fusi, Luca; Brunello, Elisabetta; Reconditi, Massimo; Piazzesi, Gabriella; Lombardi, Vincenzo

    2014-01-01

    Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre–microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4°C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa−1 (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model. PMID:24344166

  12. Engineering controllable bidirectional molecular motors based on myosin

    NASA Astrophysics Data System (ADS)

    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.

  13. Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

    PubMed Central

    Giuliano, K A; Kolega, J; DeBiasio, R L; Taylor, D L

    1992-01-01

    The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors. Images PMID:1421576

  14. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

    PubMed Central

    Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

    2015-01-01

    The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

  15. Insights into the Chemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism

    SciTech Connect

    Schwarzl, S.M.; Smith, Jeremy C; Fischer, S.

    2006-03-01

    The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP{circle_dot}Pi state of nucleotide-bound myosin.

  16. Single-molecule studies of unconventional motor protein myosin VI

    NASA Astrophysics Data System (ADS)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  17. Arabidopsis myosin XI: a motor rules the tracks.

    PubMed

    Cai, Chao; Henty-Ridilla, Jessica L; Szymanski, Daniel B; Staiger, Christopher J

    2014-11-01

    Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change. PMID:25237128

  18. Electrostatic origin of the unidirectionality of walking myosin V motors.

    PubMed

    Mukherjee, Shayantani; Warshel, Arieh

    2013-10-22

    Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ∼2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors. PMID:24106304

  19. Myosin-I molecular motors at a glance.

    PubMed

    McIntosh, Betsy B; Ostap, E Michael

    2016-07-15

    Myosin-I molecular motors are proposed to play various cellular roles related to membrane dynamics and trafficking. In this Cell Science at a Glance article and the accompanying poster, we review and illustrate the proposed cellular functions of metazoan myosin-I molecular motors by examining the structural, biochemical, mechanical and cell biological evidence for their proposed molecular roles. We highlight evidence for the roles of myosin-I isoforms in regulating membrane tension and actin architecture, powering plasma membrane and organelle deformation, participating in membrane trafficking, and functioning as a tension-sensitive dock or tether. Collectively, myosin-I motors have been implicated in increasingly complex cellular phenomena, yet how a single isoform accomplishes multiple types of molecular functions is still an active area of investigation. To fully understand the underlying physiology, it is now essential to piece together different approaches of biological investigation. This article will appeal to investigators who study immunology, metabolic diseases, endosomal trafficking, cell motility, cancer and kidney disease, and to those who are interested in how cellular membranes are coupled to the underlying actin cytoskeleton in a variety of different applications. PMID:27401928

  20. Optical trapping studies of acto-myosin motor proteins

    NASA Astrophysics Data System (ADS)

    Farrow, Rachel E.; Rosenthal, Peter B.; Mashanov, Gregory I.; Holder, Anthony A.; Molloy, Justin E.

    2007-09-01

    Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical work. This paper focuses on studies of the acto-myosin motor system that is responsible for muscle contraction and a host of other cellular motilities. Myosin works by binding to filamentous actin, pulling and then releasing. Each cycle of interaction produces a few nanometres movement and a few piconewtons force. Individual interactions can be observed directly by holding an individual actin filament between two optically trapped microspheres and positioning it in the immediate vicinity of a single myosin motor. When the chemical fuel (adenosine triphosphate or ATP) is present the myosin undergoes repeated cycles of interaction with the actin filament producing square-wave like displacements and forces. Analysis of optical trapping data sets enables the size and timing of the molecular motions to be deduced.

  1. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-01

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. A Millennial Myosin Census

    PubMed Central

    Berg, Jonathan S.; Powell, Bradford C.; Cheney, Richard E.

    2001-01-01

    The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms. PMID:11294886

  5. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2006-12-01

    Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by {approx}60 degrees. This recovery stroke is coupled to the activation of myosin's ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a see-saw motion of the relay helix, followed by a piston/seesaw motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery stroke by using Principal Component Analysis. This reveals that the only principal motions of these two helices that make a large amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions.

  6. Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

    PubMed Central

    Gabriele, Sylvain; Benoliel, Anne-Marie; Bongrand, Pierre; Théodoly, Olivier

    2009-01-01

    Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments. PMID:19450501

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

    PubMed

    Diensthuber, Ralph P; Tominaga, Motoki; Preller, Matthias; Hartmann, Falk K; Orii, Hidefumi; Chizhov, Igor; Oiwa, Kazuhiro; Tsiavaliaris, Georgios

    2015-01-01

    The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins. PMID:25326536

  9. The myosin X motor is optimized for movement on actin bundles.

    PubMed

    Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana; Blanc, Florian; Zhou, Kaifeng; Lin, Tianming; Liu, Xiaoyan; Hissier, Pascale; Samazan, Frédéric; Amigues, Béatrice; Yang, Eric D; Park, Hyokeun; Pylypenko, Olena; Cecchini, Marco; Sindelar, Charles V; Sweeney, H Lee; Houdusse, Anne

    2016-01-01

    Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles. PMID:27580874

  10. The myosin X motor is optimized for movement on actin bundles

    PubMed Central

    Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana; Blanc, Florian; Zhou, Kaifeng; Lin, Tianming; Liu, Xiaoyan; Hissier, Pascale; Samazan, Frédéric; Amigues, Béatrice; Yang, Eric D.; Park, Hyokeun; Pylypenko, Olena; Cecchini, Marco; Sindelar, Charles V.; Sweeney, H. Lee; Houdusse, Anne

    2016-01-01

    Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles. PMID:27580874

  11. Mechanics of the power stroke in myosin II

    NASA Astrophysics Data System (ADS)

    Marcucci, L.; Truskinovsky, L.

    2010-05-01

    Power stroke in skeletal muscles is a result of a conformational change in the globular portion of the molecular motor myosin II. In this paper we show that the fast tension recovery data reflecting the inner working of the power stroke mechanism can be quantitatively reproduced by a Langevin dynamics of a simple mechanical system with only two structural states. The proposed model is a generalization of the two state model of Huxley and Simmons. The main idea is to replace the rigid bistable device of Huxley and Simmons with an elastic bistable snap spring. In this setting the attached configuration of a cross bridge is represented not only by the discrete energy minima but also by a continuum of intermediate states where the fluctuation induced dynamics of the system takes place. We show that such soft-spin approach explains the load dependence of the power stroke amplitude and removes the well-known contradiction inside the conventional two state model regarding the time scale of the power stroke.

  12. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

    NASA Astrophysics Data System (ADS)

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.; Sivaramakrishnan, S.

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  13. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments.

    PubMed

    Hariadi, R F; Sommese, R F; Adhikari, A S; Taylor, R E; Sutton, S; Spudich, J A; Sivaramakrishnan, S

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes. PMID:26149240

  14. Harmonic force spectroscopy reveals a force-velocity curve from a single human beta cardiac myosin motor

    NASA Astrophysics Data System (ADS)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian; Mortensen, Kim; Flyvbjerg, Henrik; Spudich, James

    2014-03-01

    A muscle contracts rapidly under low load, but slowly under high load. Its molecular mechanisms remain to be elucidated, however. During contraction, myosins in thick filaments interact with actin in thin filaments in the sarcomere, cycling between a strongly bound (force producing) state and a weakly bound (relaxed) state. Huxley et al. have previously proposed that the transition from the strong to the weak interaction can be modulated by a load. We use a new method we call ``harmonic force spectroscopy'' to extract a load-velocity curve from a single human beta cardiac myosin II motor. With a dual-beam optical trap, we hold an actin dumbbell over a myosin molecule anchored to the microscope stage that oscillates sinusoidally. Upon binding, the motor experiences an oscillatory load with a mean that is directed forward or backward, depending on binding location We find that the bound time at saturating [ATP] is exponentially correlated with the mean load, which is explained by Arrhenius transition theory. With a stroke size measurement, we obtained a load-velocity curve from a single myosin. We compare the curves for wild-type motors with mutants that cause hypertrophic cardiomyopathies, to understand the effects on the contractile cycle

  15. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor.

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-03-01

    Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60{sup o}. This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motion of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.

  16. HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity.

    PubMed

    Samant, Sadhana A; Courson, David S; Sundaresan, Nagalingam R; Pillai, Vinodkumar B; Tan, Minjia; Zhao, Yingming; Shroff, Sanjeev G; Rock, Ronald S; Gupta, Mahesh P

    2011-02-18

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

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

    PubMed

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

    2012-08-10

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

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

    PubMed

    Jana, Biman; Onuchic, José N

    2016-08-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

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

    PubMed Central

    Jana, Biman; Onuchic, José N.

    2016-01-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

  20. Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

    PubMed Central

    Nelson, Shane R.; Trybus, Kathleen M.; Warshaw, David M.

    2014-01-01

    Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm2), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track. PMID:25201964

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

    PubMed

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

    2014-12-20

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

  2. Phospholipid-dependent regulation of the motor activity of myosin X.

    PubMed

    Umeki, Nobuhisa; Jung, Hyun Suk; Sakai, Tsuyoshi; Sato, Osamu; Ikebe, Reiko; Ikebe, Mitsuo

    2011-06-12

    Myosin X is involved in the reorganization of the actin cytoskeleton and protrusion of filopodia. Here we studied the molecular mechanism by which bovine myosin X is regulated. The globular tail domain inhibited the motor activity of myosin X in a Ca(2+)-independent manner. Structural analysis revealed that myosin X is monomeric and that the band 4.1-ezrin-radixin-moesin (FERM) and pleckstrin homology (PH) domains bind to the head intramolecularly, forming an inhibited conformation. Binding of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P(3)) to the PH domain reversed the tail-induced inhibition and induced the formation of myosin X dimers. Consistently, disruption of the binding of PtdIns(3,4,5)P(3) attenuated the translocation of myosin X to filopodial tips in cells. We propose the following mechanism: first, the tail inhibits the motor activity of myosin X by intramolecular head-tail interactions to form the folded conformation; second, phospholipid binding reverses the inhibition and disrupts the folded conformation, which induces dimer formation, thereby activating the mechanical and cargo transporter activity of myosin X.

  3. Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization

    PubMed Central

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel H.; 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. PMID:25641802

  4. Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

    PubMed

    Kengyel, András; Bécsi, Bálint; Kónya, Zoltán; Sellers, James R; Erdődi, Ferenc; Nyitrai, Miklós

    2015-05-01

    The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

  5. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.

    PubMed

    Heaslip, Aoife T; Leung, Jacqueline M; Carey, Kimberly L; Catti, Federica; Warshaw, David M; Westwood, Nicholas J; Ballif, Bryan A; Ward, Gary E

    2010-01-15

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

  6. Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor*

    PubMed Central

    Lu, Zekuan; Ma, Xiao-Nan; Zhang, Hai-Man; Ji, Huan-Hong; Ding, Hao; Zhang, Jie; Luo, Dan; Sun, Yujie; Li, Xiang-dong

    2014-01-01

    Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament. PMID:24825904

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

    PubMed Central

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

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. DOI: http://dx.doi.org/10.7554/eLife.17523.001 PMID:27623148

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

    PubMed

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

    2016-01-01

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

  9. Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

    PubMed Central

    Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2010-01-01

    Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

  10. Myosin II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

    PubMed Central

    Song, Feifei; Sang, Helen M.; Martin, René; Knölker, Hans-Joachim; MacDonald, Michael P; Weijer, Cornelis J

    2016-01-01

    Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables. PMID:25812521

  11. A New Role for Myosin II in Vesicle Fission

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

    2014-01-01

    An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis. PMID:24959909

  12. An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

    PubMed Central

    Elangovan, R; Capitanio, M; Melli, L; Pavone, F S; Lombardi, V; Piazzesi, G

    2012-01-01

    A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4–30°C) and substrate concentration. VF was 8.88 ± 0.26 μm s−1 at 30.6°C and decreased to 1.60 ± 0.09 μm s−1 at 4.5°C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40–50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5°C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads. PMID:22199170

  13. Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis.

    PubMed

    Kerridge, Stephen; Munjal, Akankshi; Philippe, Jean-Marc; Jha, Ankita; de las Bayonas, Alain Garcia; Saurin, Andrew J; Lecuit, Thomas

    2016-03-01

    Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators. PMID:26780298

  14. The myofilament elasticity and its effect on kinetics of force generation by the myosin motor.

    PubMed

    Piazzesi, Gabriella; Dolfi, Mario; Brunello, Elisabetta; Fusi, Luca; Reconditi, Massimo; Bianco, Pasquale; Linari, Marco; Lombardi, Vincenzo

    2014-06-15

    The half-sarcomere is the functional unit of striated muscle, in which, according to a "linear" mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance Cf. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (Chs-T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a Cf of 12.6nm/MPa. With this value of Cf, the fit of Chs-T relation during the isometric force rise gives s=1.8-1.9nm, similar to the values estimated using the linear model. PMID:24631572

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

    PubMed

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

    2015-04-01

    Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

  16. Molecular genetics of myosin motors in Arabidopsis. Final report, July 1, 1992--June 30, 1996

    SciTech Connect

    Schiefelbein, J.

    1997-02-01

    The normal growth and development of plant cells depends on the precise organization and distribution of the cellular contents. The basic goal of this investigation was to define a group of the molecules that are involved in organizing and transporting plant cell components. Based largely on studies of animal and fungal cells, one of the molecules thought to be involved in intracellular trafficking in plants is the actin-based motor protein myosin. Therefore, the major aim of this study was to isolate and analyze plant genes encoding myosin proteins. The plant of choice for these experiments was Arabidopsis thaliana, which offers numerous advantages for molecular genetics research.

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

    PubMed

    Nakajima, Hiroyuki; Tanoue, Takuji

    2010-02-15

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

  18. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    PubMed

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  19. Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity*

    PubMed Central

    Chinthalapudi, Krishna; Taft, Manuel H.; Martin, René; Heissler, Sarah M.; Preller, Matthias; Hartmann, Falk K.; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O.; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M.; Manstein, Dietmar J.

    2011-01-01

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway. PMID:21680745

  20. Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

    PubMed

    Chinthalapudi, Krishna; Taft, Manuel H; Martin, René; Heissler, Sarah M; Preller, Matthias; Hartmann, Falk K; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M; Manstein, Dietmar J

    2011-08-26

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

  1. Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

    SciTech Connect

    Schewkunow, Vitali; Sharma, Karan P.; Diez, Gerold; Klemm, Anna H.; Sharma, Pal C.; Goldmann, Wolfgang H.

    2008-02-08

    A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 deg. C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.

  2. Unique sequences and predicted functions of myosins in Tetrahymena thermophila.

    PubMed

    Sugita, Maki; Iwataki, Yoshinori; Nakano, Kentaro; Numata, Osamu

    2011-07-01

    Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.

  3. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

    PubMed

    Samant, Sadhana A; Pillai, Vinodkumar B; Sundaresan, Nagalingam R; Shroff, Sanjeev G; Gupta, Mahesh P

    2015-06-19

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

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

    PubMed

    Chang, Ching-Wei; Kumar, Sanjay

    2015-09-04

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.

  5. Supervillin binding to myosin II and synergism with anillin are required for cytokinesis.

    PubMed

    Smith, Tara C; Fridy, Peter C; Li, Yinyin; Basil, Shruti; Arjun, Sneha; Friesen, Ryan M; Leszyk, John; Chait, Brian T; Rout, Michael P; Luna, Elizabeth J

    2013-12-01

    Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II-binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831-1281, which bind central spindle proteins, and residues 1-170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis. PMID:24088567

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

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    2015-02-01

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

  8. SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.

    PubMed

    Kojima, S; Fujiwara, K; Onishi, H

    1999-09-01

    To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.

  9. Three-dimensional Patterns and Redistribution of Myosin II and Actin in Mitotic Dictyostelium Cells

    PubMed Central

    Neujahr, Ralph; Heizer, Christina; Albrecht, Richard; Ecke, Maria; Schwartz, Jean-Marc; Weber, Igor; Gerisch, Günther

    1997-01-01

    Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123–137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086–1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081–1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328–331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein–actin fusion. PMID:9412473

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

    PubMed Central

    Logvinova, Daria S.; Markov, Denis I.; Nikolaeva, Olga P.; Sluchanko, Nikolai N.; Ushakov, Dmitry S.; Levitsky, Dmitrii I.

    2015-01-01

    Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle. PMID:26356744

  11. Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

    PubMed Central

    Durrbach, A; Collins, K; Matsudaira, P; Louvard, D; Coudrier, E

    1996-01-01

    Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692943

  12. Dictyostelium myosin II G680V suppressors exhibit overlapping spectra of biochemical phenotypes including facilitated phosphate release.

    PubMed Central

    Wu, Y; Nejad, M; Patterson, B

    1999-01-01

    We have biochemically characterized 13 intragenic suppressors of the G680V mutation of Dictyostelium myosin II. In the absence of the G680V mutation, the suppressors result in a number of deviant behaviors, most commonly an increase in the basal (actin-independent) ATPase of the motor. This phenotype is complementary to that of the G680V mutant and supports our proposal that the latter impairs phosphate release. Different subsets of the mutants also suffer from poor ATPase enhancement by 1 mg/ml actin, failure to release from actin in the presence of ATPgammaS (or ADP and salt), and excessive release from actin in the presence of ADP. The patterns of suppressor behaviors suggest that, in general, they are facilitating P(i)-releasing state(s) of the motor, but that different individual suppressors may secondarily perturb other states or actions of the motor. PMID:10471704

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

    PubMed

    Betapudi, Venkaiah; Egelhoff, Thomas T

    2009-12-01

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

  14. Neural crest specification by inhibition of the ROCK/Myosin II pathway

    PubMed Central

    Kim, Kyeongmi; Ossipova, Olga; Sokol, Sergei Y.

    2015-01-01

    Neural crest is a population of multipotent progenitor cells that form at the border of neural and non-neural ectoderm in vertebrate embryos, and undergo epithelialmesenchymal transition and migration. According to the traditional view, the neural crest is specified in early embryos by signaling molecules including BMP, FGF and Wnt proteins. Here we identify a novel signaling pathway leading to neural crest specification, which involves Rho-associated kinase (ROCK) and its downstream target non-muscle Myosin II. We show that ROCK inhibitors promote differentiation of human embryonic stem cells into neural crest-like progenitors (NCPs) that are characterized by specific molecular markers and ability to differentiate into multiple cell types, including neurons, chondrocytes, osteocytes and smooth muscle cells. Moreover, inhibition of Myosin II was sufficient for generating NCPs at high efficiency. Whereas Myosin II has been previously implicated in the self-renewal and survival of human pluripotent ES cells, we demonstrate its role in neural crest development during ES cell differentiation. Inhibition of this pathway in Xenopus embryos expanded neural crest in vivo, further indicating that neural crest specification is controlled by ROCK-dependent Myosin II activity. We propose that changes in cell morphology in response to ROCK and Myosin II inhibition initiate mechanical signaling leading to neural crest fates. PMID:25346532

  15. Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

    In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles. PMID:18799629

  16. Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI

    PubMed Central

    Song, Chun Feng; Sader, Kasim; White, Howard; Kendrick-Jones, John; Trinick, John

    2010-01-01

    We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca2+). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO4, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix. PMID:21081082

  17. Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity.

    PubMed

    Ito, Kohji; Kashiyama, Taku; Shimada, Kiyo; Yamaguchi, Akira; Awata, Jun ya; Hachikubo, You; Manstein, Dietmar J; Yamamoto, Keiichi

    2003-12-26

    The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.

  18. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.

  19. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro. PMID:25403653

  20. Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells

    PubMed Central

    Han, Young-Eun; Lim, Ajin; Park, Sun-Hyun; Chang, Sunghoe; Lee, Suk-Ho; Ho, Won-Kyung

    2015-01-01

    AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic β-cells. PMID:26471000

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  2. Small molecule-mediated refolding and activation of myosin motor function

    PubMed Central

    Radke, Michael B; Taft, Manuel H; Stapel, Britta; Hilfiker-Kleiner, Denise; Preller, Matthias; Manstein, Dietmar J

    2014-01-01

    The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001 PMID:24520162

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

    PubMed

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

    2015-08-06

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

  4. Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I myosin.

    PubMed

    Fujita-Becker, Setsuko; Dürrwang, Ulrike; Erent, Muriel; Clark, Richard J; Geeves, Michael A; Manstein, Dietmar J

    2005-02-18

    Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.

  5. Multicomponent Analysis of Junctional Movements Regulated by Myosin II Isoforms at the Epithelial Zonula Adherens

    PubMed Central

    Smutny, Michael; Wu, Selwin K.; Gomez, Guillermo A.; Mangold, Sabine; Yap, Alpha S.; Hamilton, Nicholas A.

    2011-01-01

    The zonula adherens (ZA) of epithelial cells is a site of cell-cell adhesion where cellular forces are exerted and resisted. Increasing evidence indicates that E-cadherin adhesion molecules at the ZA serve to sense force applied on the junctions and coordinate cytoskeletal responses to those forces. Efforts to understand the role that cadherins play in mechanotransduction have been limited by the lack of assays to measure the impact of forces on the ZA. In this study we used 4D imaging of GFP-tagged E-cadherin to analyse the movement of the ZA. Junctions in confluent epithelial monolayers displayed prominent movements oriented orthogonal (perpendicular) to the ZA itself. Two components were identified in these movements: a relatively slow unidirectional (translational) component that could be readily fitted by least-squares regression analysis, upon which were superimposed more rapid oscillatory movements. Myosin IIB was a dominant factor responsible for driving the unilateral translational movements. In contrast, frequency spectrum analysis revealed that depletion of Myosin IIA increased the power of the oscillatory movements. This implies that Myosin IIA may serve to dampen oscillatory movements of the ZA. This extends our recent analysis of Myosin II at the ZA to demonstrate that Myosin IIA and Myosin IIB make distinct contributions to junctional movement at the ZA. PMID:21799860

  6. Myosin VI as a transporter and an anchor: A model for kinetics of the motor under load

    NASA Astrophysics Data System (ADS)

    Chuan, Peiying; Spudich, James; Dunn, Alexander

    2010-03-01

    Myosin VI is an actin-based motor that is thought to function both as a transporter and an anchor in vivo. In an earlier study (Altman et al, Cell 2004), inhibition of myosin VI stepping kinetics by load applied using an optical trap was observed at saturating ATP and low ADP concentrations (< 2.5 μM). A simple mechanism whereby the rate of ADP binding increases exponentially with load was proposed. This model predicts that myosin VI functions primarily as an anchor at loads greater than ˜0.5 pN under physiological nucleotide conditions, which is potentially inconsistent with its roles in vivo. Here we present myosin VI stepping data taken at a variety of applied loads and ADP concentrations, and show that the Altman model only holds at low ADP concentrations. At higher, physiologically relevant ADP concentrations under load we observe dwell times that are an order of magnitude smaller than predicted by the Altman model. We present a modified model in which applied load alters the equilibrium between two myosin VI states with different nucleotide affinities. This new kinetic scheme accurately describes myosin VI behavior at various nucleotide conditions under a large range of loads, and explains how the motor is able to carry out its roles in vivo, both as a force-generating transporter and as an anchor.

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

    PubMed Central

    Ono, Kanako; Ono, Shoichiro

    2016-01-01

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

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

    PubMed Central

    Newell-Litwa, Karen A.; Horwitz, Rick; Lamers, Marcelo L.

    2015-01-01

    ABSTRACT 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. PMID:26542704

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

    PubMed

    Newell-Litwa, Karen A; Horwitz, Rick; Lamers, Marcelo L

    2015-12-01

    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.

  10. Myosin II regulates actin rearrangement-related structural synaptic plasticity during conditioned taste aversion memory extinction.

    PubMed

    Bi, Ai-Ling; Wang, Yue; Zhang, Shuang; Li, Bo-Qin; Sun, Zong-Peng; Bi, Hong-Sheng; Chen, Zhe-Yu

    2015-03-01

    Similar to memory formation, memory extinction is also a new learning process that requires synaptic plasticity. Actin rearrangement is fundamental for synaptic plasticity, however, whether actin rearrangement in the infralimbic cortex (IL) plays a role in memory extinction, as well as the mechanisms underlying it, remains unclear. Here, using a conditioned taste aversion (CTA) paradigm, we demonstrated increased synaptic density and actin rearrangement in the IL during the extinction of CTA. Targeted infusion of an actin rearrangement inhibitor, cytochalasin D, into the IL impaired memory extinction and de novo synapse formation. Notably, we also found increased myosin II phosphorylation in the IL during the extinction of CTA. Microinfusion of a specific inhibitor of the myosin II ATPase, blebbistatin (Blebb), into the IL impaired memory extinction as well as the related actin rearrangement and changes in synaptic density. Moreover, the extinction deficit and the reduction of synaptic density induced by Blebb could be rescued by the actin polymerization stabilizer jasplakinolide (Jasp), suggesting that myosin II acts via actin filament polymerization to stabilize synaptic plasticity during the extinction of CTA. Taken together, we conclude that myosin II may regulate the plasticity of actin-related synaptic structure during memory extinction. Our studies provide a molecular mechanism for understanding the plasticity of actin rearrangement-associated synaptic structure during memory extinction.

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

    PubMed Central

    Yumura, Shigehiko; Hashima, Sayaka; Muranaka, Satsuki

    2014-01-01

    ABSTRACT Cells are always subjected to mechanical stresses, resulting in wounds of the cell membrane, but cells are able to repair and reseal their wounded membrane. Previous reports have shown that actin and myosin II accumulate around the wound and that the constriction of this purse-string closes the membrane pore. Here, we developed a microsurgical wound assay to assess wound repair in Dictyostelium cells. Fluorescent dye that had been incorporated into the cells leaked out for only 2–3 sec after wounding, and a GFP-derived, fluorescent Ca2+ sensor showed that intracellular Ca2+ transiently increased immediately after wounding. In the absence of external Ca2+, the cell failed to repair itself. During the repair process, actin accumulated at the wounded sites but myosin II did not. The wounds were repaired even in myosin II null cells to a comparable degree as the wild-type cells, suggesting that myosin II does not contribute to wound repair. Thus, the actomyosin purse-string constriction model is not a common mechanism for wound repair in eukaryotic cells, and this discrepancy may arise from the difference in cell size. PMID:25238760

  12. Non-muscle myosin II takes centre stage in cell adhesion and migration

    PubMed Central

    Vicente-Manzanares, Miguel; Ma, Xuefei; Adelstein, Robert S.; Horwitz, Alan Rick

    2010-01-01

    Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases. PMID:19851336

  13. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty; Patel, Yashomati M.

    2008-10-15

    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.

  14. Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

    PubMed Central

    Caremani, Marco; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo; Linari, Marco

    2015-01-01

    The chemo-mechanical cycle of the myosin II–actin reaction in situ has been investigated in Ca2+-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi), shortening at V ≤ 350 nm s–1 per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mm, which reduces T0 and the number of motors by 40–50%, does not influence the dependence on V of number and strain. A model simulation of the myosin–actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. Key points Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin–actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to

  15. Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii.

    PubMed

    Kuznicki, J; Atkinson, M A; Korn, E D

    1984-07-25

    Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed. PMID:6235225

  16. Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.

    PubMed

    Trivedi, Darshan V; David, Charles; Jacobs, Donald J; Yengo, Christopher M

    2012-06-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.

  17. Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V

    PubMed Central

    Trivedi, Darshan V.; David, Charles; Jacobs, Donald J.; Yengo, Christopher M.

    2012-01-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region. PMID:22713570

  18. Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

    PubMed

    Zhang, Ying; Kawamichi, Hozumi; Tanaka, Hideyuki; Yoshiyama, Shinji; Kohama, Kazuhiro; Nakamura, Akio

    2012-08-01

    We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

  19. Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

    PubMed

    Mizutani, Takeomi; Li, Rui; Haga, Hisashi; Kawabata, Kazushige

    2015-09-18

    The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

  20. Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells

    PubMed Central

    Moore, Cathy C; Lakner, Ashley M; Yengo, Christopher M; Schrum, Laura W

    2011-01-01

    AIM: To identify and characterize the function of nonmuscle myosin II (NMM II) isoforms in primary rat hepatic stellate cells (HSCs). METHODS: Primary HSCs were isolated from male Sprague-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM II isoform (II-A, II-B and II-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM II protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM IIisoform expression determined by immunohistochemistry. Using a selective myosin II inhibitor and siRNA-mediated knockdown of each isoform, NMM II functionality in primary rat HSCs was determined by contraction and migration assays. RESULTS: NMM II-A and II-B mRNA expression was increased in culture-activated HSCs (Day 14) with significant increases seen in all pair-wise comparisons (II-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; II-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (II-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; II-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No significant differences were observed in NMM II-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM II-A and II-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro studies showed increased expression of NMM II-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM II-A and II-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM II-A and II-B to migration and contraction, NMM II-A and II-B expression were downregulated with siRNA. NMM II

  1. Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle.

    PubMed

    Trivedi, Darshan V; Muretta, Joseph M; Swenson, Anja M; Thomas, David D; Yengo, Christopher M

    2013-07-01

    We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

  2. Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance.

    PubMed

    Jacot, Damien; Daher, Wassim; Soldati-Favre, Dominique

    2013-06-12

    Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

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

  6. Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis

    PubMed Central

    Spudich, James A.; Sivaramakrishnan, Sivaraj

    2010-01-01

    The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual ~ 180° lever arm swing and achieves its large step size using special structural features in its tail domain. PMID:20094053

  7. New insights into myosin evolution and classification.

    PubMed

    Foth, Bernardo J; Goedecke, Marc C; Soldati, Dominique

    2006-03-01

    Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

  8. Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility

    PubMed Central

    Hanley, Peter J.; Xu, Yan; Kronlage, Moritz; Grobe, Kay; Schön, Peter; Song, Jian; Sorokin, Lydia; Schwab, Albrecht; Bähler, Martin

    2010-01-01

    Directional motility is a fundamental function of immune cells, which are recruited to sites of pathogen invasion or tissue damage by chemoattractant signals. To move, cells need to generate lamellipodial membrane protrusions at the front and retract the trailing end. These elementary events are initiated by Rho-family GTPases, which cycle between active GTP-bound and inactive GDP-bound states. How the activity of these “molecular switches” is spatially coordinated is only beginning to be understood. Here, we show that myosin IXb (Myo9b), a Rho GTPase-activating protein (RhoGAP) expressed in immune cells, is essential for coordinating the activity of Rho. We generated Myo9b-deficient mice and show that Myo9b−/− macrophages have strikingly defective spreading and polarization. Furthermore, Myo9b−/− macrophages fail to generate lamellipodia in response to a chemoattractant, and migration in a chemotactic gradient is severely impaired. Inhibition of Rho rescues the Myo9b−/− phenotype, but impairs tail retraction. We also found that Myo9b is important in vivo. Chemoattractant-induced monocyte recruitment to the peritoneal cavity is substantially reduced in Myo9b−/− mice. Thus, we identify the “motorized Rho inhibitor” Myo9b as a key molecular component required for spatially coordinated cell shape changes and motility. PMID:20566876

  9. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

    PubMed Central

    Ebrahim, Seham; Avenarius, Matthew R.; Grati, M'hamed; Krey, Jocelyn F.; Windsor, Alanna M.; Sousa, Aurea D.; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A.; Salles, Felipe T.; Baird, Michelle A.; Davidson, Michael W.; Jones, Sherri M.; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H.; Yengo, Christopher M.; Barr-Gillespie, Peter G.; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

  10. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

    PubMed

    Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed; Krey, Jocelyn F; Windsor, Alanna M; Sousa, Aurea D; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A; Salles, Felipe T; Baird, Michelle A; Davidson, Michael W; Jones, Sherri M; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H; Yengo, Christopher M; Barr-Gillespie, Peter G; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

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

    SciTech Connect

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    2014-08-01

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

  12. The neck region of the myosin motor domain acts as a lever arm to generate movement.

    PubMed Central

    Uyeda, T Q; Abramson, P D; Spudich, J A

    1996-01-01

    The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8633089

  13. Myosin-II controls cellular branching morphogenesis and migration in 3D by minimizing cell surface curvature

    PubMed Central

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

    2014-01-01

    In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration. PMID:25621949

  14. Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension

    PubMed Central

    Tetley, Robert J; Blanchard, Guy B; Fletcher, Alexander G; Adams, Richard J; Sanson, Bénédicte

    2016-01-01

    Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. Here, we develop novel computational methods to analyse the spatiotemporal dynamics of Myosin II during GBE, at the scale of the tissue. We show that initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. Our results are consistent with recent work showing that a combinatorial code of Toll-like receptors downstream of pair-rule genes contributes to Myosin II polarization via local cell-cell interactions. We propose an updated cell-cell interaction model for Myosin II polarization that we tested in a vertex-based simulation. DOI: http://dx.doi.org/10.7554/eLife.12094.001 PMID:27183005

  15. Genetics Home Reference: distal hereditary motor neuropathy, type II

    MedlinePlus

    ... hereditary motor neuropathy, type II distal hereditary motor neuropathy, type II Enable Javascript to view the expand/ ... Open All Close All Description Distal hereditary motor neuropathy, type II is a progressive disorder that affects ...

  16. Cell rigidity and shape override CD47's "self"-signaling in phagocytosis by hyperactivating myosin-II.

    PubMed

    Sosale, Nisha G; Rouhiparkouhi, Tahereh; Bradshaw, Andrew M; Dimova, Rumiana; Lipowsky, Reinhard; Discher, Dennis E

    2015-01-15

    A macrophage engulfs another cell or foreign particle in an adhesive process that often activates myosin-II, unless the macrophage also engages "marker of self" CD47 that inhibits myosin. For many cell types, adhesion-induced activation of myosin-II is maximized by adhesion to a rigid rather than a flexible substrate. Here we demonstrate that rigidity of a phagocytosed cell also hyperactivates myosin-II, which locally overwhelms self-signaling at a phagocytic synapse. Cell stiffness is one among many factors including shape that changes in erythropoiesis, in senescence and in diseases ranging from inherited anemias and malaria to cancer. Controlled stiffening of normal human red blood cells (RBCs) in different shapes does not compromise CD47's interaction with the macrophage self-recognition receptor signal regulatory protein alpha (SIRPA). Uptake of antibody-opsonized RBCs is always fastest with rigid RBC discocytes, which also show that maximal active myosin-II at the synapse can dominate self-signaling by CD47. Rigid but rounded RBC stomatocytes signal self better than rigid RBC discocytes, highlighting the effects of shape on CD47 inhibition. Physical properties of phagocytic targets thus regulate self signaling, as is relevant to erythropoiesis, to clearance of rigid RBCs after blood storage, clearance of rigid pathological cells such as thalassemic or sickle cells, and even to interactions of soft/stiff cancer cells with macrophages.

  17. The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II.

    PubMed

    Rujano, Maria A; Sanchez-Pulido, Luis; Pennetier, Carole; le Dez, Gaelle; Basto, Renata

    2013-11-01

    Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. Here, we characterized the function of the Drosophila ASPM orthologue, Asp, and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. We show that size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. We propose that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness.

  18. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    PubMed

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion. PMID:15726816

  19. Myosins and cell dynamics in cellular slime molds.

    PubMed

    Yumura, Shigehiko; Uyeda, Taro Q P

    2003-01-01

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

  20. The Effect of Including the C2 Insert of Nonmuscle Myosin II-C on Neuritogenesis*

    PubMed Central

    Saha, Shekhar; Dey, Sumit K.; Biswas, Arunima; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    2013-01-01

    The functional role of the C2 insert of nonmuscle myosin II-C (NM II-C) is poorly understood. Here, we report for the first time that the expression of the C2 insert-containing isoform, NM II-C1C2, is inducible in Neuro-2a cells during differentiation both at mRNA and protein levels. Immunoblot and RT-PCR analysis reveal that expression of NM II-C1C2 peaks between days 3 and 6 of differentiation. Localization of NM II-C1C2 in Neuro-2a cells suggests that the C2 insert-containing isoform is localized in the cytosol and along the neurites, specifically at the adherence point to substratum. Inhibition of endogenous NM II-C1C2 using siRNA decreases the neurite length by 43% compared with control cells treated with nonspecific siRNA. Time lapse image analysis reveals that neurites of C2-siRNA-treated cells have a net negative change in neurite length per minute, leading to a reduction of overall neurite length. During neuritogenesis, NM II-C1C2 can interact and colocalize with β1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites. PMID:23355468

  1. Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

    PubMed Central

    Yashiro, Hanako; Loza, Andrew J.; Skeath, James B.; Longmore, Gregory D.

    2014-01-01

    Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia. PMID:25079692

  2. Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

    PubMed Central

    Ng, Mei Rosa; Besser, Achim

    2012-01-01

    The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration. PMID:23091067

  3. Nonmuscle Myosin II Regulates the Morphogenesis of Metanephric Mesenchyme-Derived Immature Nephrons.

    PubMed

    Recuenco, Mariam C; Ohmori, Tomoko; Tanigawa, Shunsuke; Taguchi, Atsuhiro; Fujimura, Sayoko; Conti, Mary Anne; Wei, Qize; Kiyonari, Hiroshi; Abe, Takaya; Adelstein, Robert S; Nishinakamura, Ryuichi

    2015-05-01

    The kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud. The mesenchyme transforms into epithelia and forms complicated nephron structures, whereas the ureteric bud extends its pre-existing epithelial ducts. Although the roles are well established for extracellular stimuli, such as Wnt and Notch, it is unclear how the intracellular cytoskeleton regulates these morphogenetic processes. Myh9 and Myh10 encode nonmuscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases and focal segmental glomerulosclerosis in adults. Here, we analyzed the roles of Myh9 and Myh10 in the developing kidney. Ureteric bud-specific depletion of Myh9 resulted in no apparent phenotypes, whereas mesenchyme-specific Myh9 deletion caused proximal tubule dilations and renal failure. Mesenchyme-specific Myh9/Myh10 mutant mice died shortly after birth and showed a severe defect in nephron formation. The nascent mutant nephrons failed to form a continuous lumen, which likely resulted from impaired apical constriction of the elongating tubules. In addition, nephron progenitors lacking Myh9/Myh10 or the possible interactor Kif26b were less condensed at midgestation and reduced at birth. Taken together, nonmuscle myosin II regulates the morphogenesis of immature nephrons derived from the metanephric mesenchyme and the maintenance of nephron progenitors. Our data also suggest that Myh9 deletion in mice results in failure to maintain renal tubules but not in glomerulosclerosis.

  4. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles.

    PubMed

    Gally, Christelle; Wissler, Frédéric; Zahreddine, Hala; Quintin, Sophie; Landmann, Frédéric; Labouesse, Michel

    2009-09-01

    Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4. PMID:19675126

  5. The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts▿ †

    PubMed Central

    Almagro, Sébastien; Durmort, Claire; Chervin-Pétinot, Adeline; Heyraud, Stephanie; Dubois, Mathilde; Lambert, Olivier; Maillefaud, Camille; Hewat, Elizabeth; Schaal, Jean Patrick; Huber, Philippe; Gulino-Debrac, Danielle

    2010-01-01

    Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings. PMID:20123970

  6. Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

    PubMed

    Sellers, James R; Wang, Fei; Chantler, Peter D

    2003-01-01

    Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

  7. Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis.

    PubMed

    Kondo, Tomo; Isoda, Rieko; Uchimura, Takashi; Sugiyama, Mutsumi; Hamao, Kozue; Hosoya, Hiroshi

    2012-01-13

    Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II. PMID:22166199

  8. Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece.

    PubMed

    Liu, Xiong; Hong, Myoung-Soon; Shu, Shi; Yu, Shuhua; Korn, Edward D

    2013-01-01

    Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain. PMID:23248285

  9. Characterization of Amoeba proteus myosin VI immunoanalog.

    PubMed

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

    2005-07-01

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

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

    PubMed Central

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

    2014-01-01

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions. PMID:24591646

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

    PubMed

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

    2014-03-18

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

  12. Myosin motor function: the ins and outs of actin-based membrane protrusions

    PubMed Central

    Nambiar, Rajalakshmi; McConnell, Russell E.

    2011-01-01

    Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion. PMID:20107861

  13. The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

    PubMed

    Tumbarello, David A; Manna, Paul T; Allen, Mark; Bycroft, Mark; Arden, Susan D; Kendrick-Jones, John; Buss, Folma

    2015-10-01

    Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.

  14. SDF-1α stiffens myeloma bone marrow mesenchymal stromal cells through the activation of RhoA-ROCK-Myosin II

    PubMed Central

    Choi, Dong Soon; Stark, Daniel J.; Raphael, Robert M.; Wen, Jianguo; Su, Jing; Zhou, Xiaobo; Chang, Chung-Che; Zu, Youli

    2015-01-01

    Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF-1α-induced activation of MYL2 through the G(i./o)-PI3K-RhoA-ROCK-Myosin II signaling pathway, affecting Young’s modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell–cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF-1α signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs. PMID:25137150

  15. Various Themes of Myosin Regulation.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2016-05-01

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

  16. An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.

    PubMed

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro. PMID:27473959

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

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

    PubMed Central

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

    2015-01-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells1,2. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems3,4. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears—speed up, slow down or switch directions—when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

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

    PubMed

    Pedersen, S F; Hoffmann, E K

    2002-07-01

    Osmotic shrinkage of Ehrlich ascites tumor cells (EATC) elicited translocation of myosin II from the cytosol to the cortical region, and swelling elicits concentration of myosin II in the Golgi region. Rho kinase and p38 both appeared to be involved in shrinkage-induced myosin II reorganization. In contrast, the previously reported shrinkage-induced actin polymerization [Pedersen et al. (1999) Exp. Cell Res. 252, 63-74] was independent of Rho kinase, p38, myosin light chain kinase (MLCK), and protein kinase C (PKC), which thus do not exert their effects on the shrinkage-activated transporters via 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 by osmotic shrinkage and by the serine/threonine phosphatase inhibitor Calyculin A (CL-A). Both stimuli caused Rho kinase-dependent myosin II relocation to the cortical cytoplasm, but in contrast to the shrinkage-induced F-actin polymerization, CL-A treatment elicited a slight F-actin depolymerization. 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. PMID:12061817

  1. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

    PubMed

    Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I; Geeves, Michael A

    2016-01-22

    The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

  2. Mesenchymal chemotaxis requires selective inactivation of myosin II at the leading edge via a noncanonical PLCγ/PKCα pathway.

    PubMed

    Asokan, Sreeja B; Johnson, Heath E; Rahman, Anisur; King, Samantha J; Rotty, Jeremy D; Lebedeva, Irina P; Haugh, Jason M; Bear, James E

    2014-12-22

    Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.

  3. Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    PubMed Central

    Arora, Sneha; Saha, Shekhar; Roy, Saheli; Das, Madhurima; Jana, Siddhartha S.

    2015-01-01

    It is the promise of regeneration and therapeutic applications that has sparked an interest in mesenchymal stem cells (MSCs). Following infusion, MSCs migrate to sites of injury or inflammation by virtue of their homing property. To exert optimal clinical benefits, systemically delivered MSCs need to migrate efficiently and in adequate numbers to pathological areas in vivo. However, underlying molecular mechanisms responsible for MSC migration are still not well understood. The Wharton's jelly (WJ) of the umbilical cord is an attractive source of MSCs for stem cell therapy because of its abundant availability and painless collection. In this study, we attempted to identify the role of nonmuscle myosin II (NMII), if any, in the migration of WJ-derived MSCs (WJ-MSCs). Expression of NMII isoforms, NMIIA, and NMIIB was observed both at RNA and protein levels in WJ-MSCs. Inhibition of NMII or its regulator ROCK, by pharmacological inhibitors, resulted in significant reduction in the migration of WJ-MSCs as confirmed by the scratch migration assay and time-lapse microscopy. Next, trying to dissect the role of each NMII isoform in migration of WJ-MSCs, we found that siRNA-mediated downregulation of NMIIA, but not NMIIB expression, led to cells failing to retract their trailing edge and losing cell–cell cohesiveness, while exhibiting a nondirectional migratory pathway. Migration, moreover, is also dependent on optimal affinity adhesion, which would allow rapid attachment and release of cells and, hence, can be influenced by extracellular matrix (ECM) and adhesion molecules. We demonstrated that inhibition of NMII and more specifically NMIIA resulted in increased gene expression of ECM and adhesion molecules, which possibly led to stronger adhesions and, hence, decreased migration. Therefore, these data suggest that NMII acts as a regulator of cell migration and adhesion in WJ-MSCs. PMID:25923805

  4. Nonmuscle myosin II powered transport of newly formed collagen fibrils at the plasma membrane

    PubMed Central

    Kalson, Nicholas S.; Starborg, Tobias; Lu, Yinhui; Mironov, Aleksandr; Humphries, Sally M.; Holmes, David F.; Kadler, Karl E.

    2013-01-01

    Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1–10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures. PMID:24248360

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

    PubMed

    Uyeda, T Q; Yumura, S

    2000-04-15

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

  6. Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

    PubMed Central

    Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo

    2015-01-01

    Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii. PMID:26544049

  7. Cortical F-actin, the exocytic mode, and neuropeptide release in mouse chromaffin cells is regulated by myristoylated alanine-rich C-kinase substrate and myosin II.

    PubMed

    Doreian, Bryan W; Fulop, Tiberiu G; Meklemburg, Robert L; Smith, Corey B

    2009-07-01

    Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.

  8. Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes*

    PubMed Central

    Bultema, Jarred J.; Boyle, Judith A.; Malenke, Parker B.; Martin, Faye E.; Dell'Angelica, Esteban C.; Cheney, Richard E.; Di Pietro, Santiago M.

    2014-01-01

    Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. PMID:25324551

  9. Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

    PubMed

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

  10. Myosin II ATPase Activity Mediates the Long-Term Potentiation-Induced Exodus of Stable F-Actin Bound by Drebrin A from Dendritic Spines

    PubMed Central

    Mizui, Toshiyuki; Sekino, Yuko; Yamazaki, Hiroyuki; Ishizuka, Yuta; Takahashi, Hideto; Kojima, Nobuhiko; Kojima, Masami; Shirao, Tomoaki

    2014-01-01

    The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca2+ influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement. PMID:24465547

  11. Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica.

    PubMed

    Coudrier, Evelyne; Amblard, François; Zimmer, Christophe; Roux, Pascal; Olivo-Marin, Jean-Christophe; Rigothier, Marie-Christine; Guillén, Nancy

    2005-01-01

    Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two-photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco-2 cell monolayers, an enterocyte-like model, and hamster liver shows that E. histolytica undergoes non-Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal-GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal-GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.

  12. Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling

    PubMed Central

    Ossipova, Olga; Kim, Kyeongmi; Sokol, Sergei Y.

    2015-01-01

    The vertebrate neural tube forms as a result of complex morphogenetic movements, which require the functions of several core planar cell polarity (PCP) proteins, including Vangl2 and Prickle. Despite the importance of these proteins for neurulation, their subcellular localization and the mode of action have remained largely unknown. Here we describe the anteroposterior planar cell polarity (AP-PCP) of the cells in the Xenopus neural plate. At the neural midline, the Vangl2 protein is enriched at anterior cell edges and that this localization is directed by Prickle, a Vangl2-interacting protein. Our further analysis is consistent with the model, in which Vangl2 AP-PCP is established in the neural plate as a consequence of Wnt-dependent phosphorylation. Additionally, we uncover feedback regulation of Vangl2 polarity by Myosin II, reiterating a role for mechanical forces in PCP. These observations indicate that both Wnt signaling and Myosin II activity regulate cell polarity and cell behaviors during vertebrate neurulation. PMID:25910938

  13. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  14. Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor

    PubMed Central

    Himmel, D. M.; Gourinath , S.; Reshetnikova, L.; Shen, Y.; Szent-Györgyi, A. G.; Cohen, C.

    2002-01-01

    Here we report a 2.3-Å crystal structure of scallop myosin S1 complexed with ADP⋅BeFx, as well as three additional structures (at 2.8–3.8 Å resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-Å scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter/lever arm module from the motor and allows cross-linking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-Å nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release. PMID:12297624

  15. She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p

    PubMed Central

    Böhl, Florian; Kruse, Claudia; Frank, Andrea; Ferring, Dunja; Jansen, Ralf-Peter

    2000-01-01

    RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences (‘zipcodes’) within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p’s C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA–motor protein adapters. PMID:11032818

  16. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  17. The Cdk5 activator P39 specifically links muskelin to myosin II and regulates stress fiber formation and actin organization in lens.

    PubMed

    Tripathi, Brajendra K; Lowy, Douglas R; Zelenka, Peggy S

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, requires p39 for its enzymatic activity, and is implicated in cytoskeletal organization and contraction in numerous cell types. The C-terminus of p39 binds muskelin, a multi-domain scaffolding protein known to affect cytoskeletal organization, but the mechanisms by which muskelin affects cytoskeletal organization remain unclear. The present study sought to determine whether p39 might serve as an adaptor protein that links muskelin to stress fibers and to investigate the possible biological relevance of such an interaction. Double immunoprecipitation showed that muskelin, p39, and myosin II are components of a single intracellular complex, and suppressing p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin II. Muskelin is colocalized with myosin regulatory light chain (MRLC) and on stress fibers. The suppression of muskelin reduced Rho-GTP, MRLC phosphorylation, disrupted stress fiber organization, and promoted cell migration, all of which closely mimic the effect of Cdk5 inhibition. Moreover, suppressing muskelin and inhibiting Cdk5 together have no additional effect, indicating that muskelin plays an important role in Cdk5-dependent signaling. p39 is necessary and sufficient for Cdk5-dependent regulation of MRLC phosphorylation, as suppression of p39, but not p35, reduces MRLC phosphorylation. Together, these results demonstrate that p39 specifically links muskelin to myosin II and consequently, to stress fibers and reveal a novel role for muskelin in regulating myosin phosphorylation and cytoskeletal organization. PMID:25128817

  18. The Cdk5 activator P39 specifically links muskelin to myosin II and regulates stress fiber formation and actin organization in lens.

    PubMed

    Tripathi, Brajendra K; Lowy, Douglas R; Zelenka, Peggy S

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, requires p39 for its enzymatic activity, and is implicated in cytoskeletal organization and contraction in numerous cell types. The C-terminus of p39 binds muskelin, a multi-domain scaffolding protein known to affect cytoskeletal organization, but the mechanisms by which muskelin affects cytoskeletal organization remain unclear. The present study sought to determine whether p39 might serve as an adaptor protein that links muskelin to stress fibers and to investigate the possible biological relevance of such an interaction. Double immunoprecipitation showed that muskelin, p39, and myosin II are components of a single intracellular complex, and suppressing p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin II. Muskelin is colocalized with myosin regulatory light chain (MRLC) and on stress fibers. The suppression of muskelin reduced Rho-GTP, MRLC phosphorylation, disrupted stress fiber organization, and promoted cell migration, all of which closely mimic the effect of Cdk5 inhibition. Moreover, suppressing muskelin and inhibiting Cdk5 together have no additional effect, indicating that muskelin plays an important role in Cdk5-dependent signaling. p39 is necessary and sufficient for Cdk5-dependent regulation of MRLC phosphorylation, as suppression of p39, but not p35, reduces MRLC phosphorylation. Together, these results demonstrate that p39 specifically links muskelin to myosin II and consequently, to stress fibers and reveal a novel role for muskelin in regulating myosin phosphorylation and cytoskeletal organization.

  19. Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin.

    PubMed

    Iwai, Sosuke; Chaen, Shigeru

    2007-05-25

    The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

  20. Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

    PubMed Central

    Kondo, Tomo; Isoda, Rieko; Ookusa, Takayuki; Kamijo, Keiju; Hamao, Kozue; Hosoya, Hiroshi

    2013-01-01

    Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells. PMID:23951055

  1. Evidence that Myosin Activity Opposes Microtubule-based Axonal Transport of Mitochondria

    PubMed Central

    Pathak, Divya; Sepp, Katharine J.; Hollenbeck, Peter J.

    2010-01-01

    Neurons transport and position mitochondria using a combination of saltatory, bidirectional movements and stationary docking. Axonal mitochondria move along microtubules (MTs) using kinesin and dynein motors, but actin and myosin also play a poorly-defined role in their traffic. To ascertain this role, we have used RNA interference to deplete specific myosin motors in cultured Drosophila neurons and quantified the effects on mitochondrial motility. We produced a fly strain expressing the C. elegans RNA transporter SID-1 in neurons to increase the efficacy of RNAi in primary cultures. These neurons exhibited significantly increased RNAi-mediated knockdown of gene expression compared to neurons not expressing this transporter. Using this system, we observed a significant increase in mitochondrial transport upon myosin V depletion. Mitochondrial mean velocity and duty cycle were augmented in both anterograde and retrograde directions, and the fraction of mitochondrial flux contained in long runs almost doubled for anterograde movement. Myosin VI depletion increased the same movement parameters, but was selective for retrograde movement, while myosin II depletion produced no phenotype. An additional effect of myosin V depletion was an increase in mitochondrial length. These data indicate that myosin V and VI play related but distinct roles in regulating MT-based mitochondrial movement: they oppose, rather than complement protracted MT-based movements and perhaps facilitate organelle docking. PMID:20592219

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. Effects of Alcohol, Lithium, and Homocysteine on Nonmuscle Myosin-II in the Mouse Placenta and Human Trophoblasts

    PubMed Central

    HAN, Mingda; NEVES, Ana Luisa; SERRANO, Maria; BRINEZ, Pilar; HUHTA, James C.; ACHARYA, Ganesh; LINASK, Kersti K.

    2012-01-01

    Mouse embryonic exposure to alcohol, lithium, and homocysteine results in intrauterine growth restriction (IUGR) and cardiac defects. Our present study focuses on the placental effects. Objectives We analyzed the hypothesis that expression of nonmuscle myosin (NMM)-II isoforms involved in cell motility, mechanosensing, and extracellular matrix assembly, are altered by the three factors in human trophoblast (HTR8/SVneo) cells in vitro and in the mouse placenta in vivo. Study Design After exposure during gastrulation to alcohol, homocysteine, or lithium, ultrasonography defined embryos exhibiting abnormal placental blood flow. Results NMM-IIA /NMM-IIB are differentially expressed in trophoblasts and in mouse placental vascular endothelial cells under pathological conditions. Misexpression of NMM-IIA/ NMM-IIB in the affected placentas continued stably to mid-gestation, but can be prevented by folate and myo-inositol supplementation. Conclusions It is concluded that folate and myo-inositol initiated early in mouse pregnancy can restore NMM-II expression, permit normal placentation/embryogenesis, and prevent IUGR induced by alcohol, lithium, and homocysteine. PMID:22704764

  6. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Day, I. S.

    2001-01-01

    BACKGROUND: Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. RESULTS: Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. CONCLUSIONS: Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

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

    PubMed Central

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

    2009-01-01

    SUMMARY The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ~9 nm distance between the myosin lever arm and the thin filament. In order to localize the N-terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle. PMID:17597155

  8. Small-molecule inhibitors of myosin proteins

    PubMed Central

    Bond, Lisa M; Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2014-01-01

    Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design. PMID:23256812

  9. Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins.

    PubMed

    Seki, Masaya; Kashiyama, Taku; Hachikubo, You; Ito, Kohji; Yamamoto, Keiichi

    2004-11-19

    A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.

  10. F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

    PubMed

    Villanueva, José; Torres, Vanesa; Torregrosa-Hetland, Cristina J; Garcia-Martinez, Virginia; López-Font, Inmaculada; Viniegra, Salvador; Gutiérrez, Luis M

    2012-10-01

    Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.

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

    PubMed Central

    Sabbir, Mohammad G.; Dillon, Rachelle; Mowat, Michael R. A.

    2016-01-01

    ABSTRACT The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  12. Epidermal Growth Factor Signalling Controls Myosin II Planar Polarity to Orchestrate Convergent Extension Movements during Drosophila Tubulogenesis

    PubMed Central

    Bunt, Stephanie; Bischoff, Marcus; VijayRaghavan, Krishnaswamy; Skaer, Helen

    2014-01-01

    Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis we show that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, we demonstrate that the vector of cell movement is regulated by localised epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality. PMID:25460353

  13. Directional Mechanosensing in Myosin VI

    NASA Astrophysics Data System (ADS)

    Yang, Yubo; Tehver, Riina

    2013-03-01

    Myosin is a family of versatile motor proteins that perform various tasks, such as organelle transport, anchoring and cell deformation. Although the general mechanism of the motors has been fairly well established, details on dynamic aspects like force response of the motor, and force propagation are yet to be fully understood. In this poster, we present the response of the ATP binding region to force exerted on the tail domain in order to test the proposed tension-dependent gating mechanism of myosin VI processive motion. We employed the Self-Organized Polymer model in a computer simulation to explore the effect. Current results show that the ATP binding domain of myosin VI indeed exhibits tension dependence - both structurally and dynamically.

  14. Two-headed binding of a processive myosin to F-actin.

    PubMed

    Walker, M L; Burgess, S A; Sellers, J R; Wang, F; Hammer, J A; Trinick, J; Knight, P J

    2000-06-15

    Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation. PMID:10866203

  15. Molecular engineering of myosin.

    PubMed Central

    Manstein, Dietmar J

    2004-01-01

    Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins. PMID:15647166

  16. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

  17. Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

    PubMed

    Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

    2013-03-01

    In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

  18. Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

    PubMed Central

    Bi, Guo-Qiang; Morris, Robert L.; Liao, Guochun; Alderton, Janet M.; Scholey, Jonathan M.; Steinhardt, Richard A.

    1997-01-01

    Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain. PMID:9281579

  19. Myosins in protists.

    PubMed

    Gavin, R H

    2001-01-01

    This review focuses on selected papers that illustrate an historical perspective and the current knowledge of myosin structure and function in protists. The review contains a general description of myosin structure, a phylogenetic tree of the myosin classes, and descriptions of myosin isoforms identified in protists. Each myosin is discussed within the context of the taxonomic group of the organism in which the myosin has been identified. Domain structure, cellular location, function, and regulation are described for each myosin.

  20. A Role for Myosin V Motor Proteins in the Selective Delivery of Kv Channel Isoforms to the Membrane Surface of Cardiac Myocytes

    PubMed Central

    Schumacher-Bass, Sarah M.; Vesely, Eileen D.; Zhang, Lian; Ryland, Katherine E.; McEwen, Dyke P.; Chan, Priscilla J.; Frasier, Chad R.; McIntyre, Jeremy C.; Shaw, Robin M.; Martens, Jeffrey R.

    2014-01-01

    Rationale Kv1.5 (KCNA5) mediates the IKur current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation (AF), Kv1.5 has emerged as a promising target for the treatment of AF. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. Objective To investigate the role of the unconventional myosin V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. Methods and Results Western Blot analysis showed MYO5A and MYO5B expression in the heart, while disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and shRNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and Connexin-43 (Cx43), but not hERG (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, while MYO5B regulated post-endocytic recycling. Over-expression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. Conclusions MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current, and consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias. PMID:24508725

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

    PubMed

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

    2007-04-15

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

  2. [Role of myosins in depression of sensitivity of Helix neurons to acetylcholine in a cellular analog of habituation].

    PubMed

    Pivovarov, A S; Murzina, G B; Makhnovskiĭ, D A; Vasil'eva, N A; Tret'iakova, M S

    2013-01-01

    We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

  3. A mechanochemical model for myosin VI

    NASA Astrophysics Data System (ADS)

    Tehver, Riina; Jack, Amanda; Lowe, Ian

    Myosin VI is a motor protein that transports cellular cargo along actin filaments. This transport takes place as a result of a coordinated mechano-chemical cycle that is controlled by external variables including imposed force and nucleotide concentrations. We present a model that captures the different dynamic pathways that myosin VI can take in response to these variables. The results of our model for experimentally observable quantities, such as the motor velocity or run length, agree with available experimental data, and we can also make predictions beyond the tested regimes. Using the model, we study how myosin VI reacts to its environment and test its operational efficiency.

  4. The RhoA-Rok-Myosin II Pathway is Involved in Extracellular Matrix-Mediated Regulation of Prolactin Signaling in Mammary Epithelial Cells

    PubMed Central

    Du, Jyun-Yi; Chen, Meng-Chi; Hsu, Tsai-Ching; Wang, Jen-Hsing; Brackenbury, Lisa; Lin, Ting-Hui; Wu, Yi-Ying; Yang, Zhihong; Streuli, Charles H; Lee, Yi-Ju

    2012-01-01

    In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions. J. Cell. Physiol. 227: 1553–1560, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678418

  5. Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct.

    PubMed

    Chou, Chung-Lin; Yu, Ming-Jiun; Kassai, Eliza M; Morris, Ryan G; Hoffert, Jason D; Wall, Susan M; Knepper, Mark A

    2008-07-01

    Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry.

  6. The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

    PubMed

    Gandía, Mónica; Harries, Eleonora; Marcos, Jose F

    2014-06-01

    Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.

  7. Kinetic and Motor Functions Mediated by Distinct Regions of the Regulatory Light Chain of Smooth Muscle Myosin1,2

    PubMed Central

    Ni, Shaowei; Hong, Feng; Brewer, Paul D.; Ikebe, Mitsuo; Onishi, Hirofumi; Baker, Jonathan E.; Facemyer, Kevin C.; Cremo, Christine R.

    2009-01-01

    To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP95-98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg2+-ATPase (Vmax and Katpase) and in vitro actin sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the upstate (0.007-0.030 s-1) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (Katpase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the Vmax was 64% and actin sliding velocity was ∼50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation. PMID:19635597

  8. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

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

    PubMed Central

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

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

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

    PubMed

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

    2016-02-18

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

  11. The size and the speed of the working stroke of muscle myosin and its dependence on the force

    PubMed Central

    Piazzesi, Gabriella; Lucii, Leonardo; Lombardi, Vincenzo

    2002-01-01

    Myosin II is the motor protein that produces force and shortening in muscle by ATP-driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head. PMID:12433956

  12. The Effects of Extracellular Calcium on Motility, Pseudopod and Uropod Formation, Chemotaxis and the Cortical Localization of Myosin II in Dictyostelium discoideum

    PubMed Central

    Lusche, Daniel F.; Wessels, Deborah; Soll, David R.

    2009-01-01

    Extracellular Ca++, a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca++ is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca++ only affects turning, but in combination with extracellular Ca++, enhances the effects of extracellular Ca++. Potassium, at 40 mM, can substitute for Ca++. Mg++, Mn++, Zn++ and Na+ cannot. Extracellular Ca++, or K+, also induce the cortical localization of myosin II in a polar fashion. The effects of Ca++, K+ or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca++. These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. PMID:19363786

  13. All-atom molecular dynamics simulations of actin-myosin interactions: a comparative study of cardiac α myosin, β myosin, and fast skeletal muscle myosin.

    PubMed

    Li, Minghui; Zheng, Wenjun

    2013-11-26

    Myosins are a superfamily of actin-binding motor proteins with significant variations in kinetic properties (such as actin binding affinity) between different isoforms. It remains unknown how such kinetic variations arise from the structural and dynamic tuning of the actin-myosin interface at the amino acid residue level. To address this key issue, we have employed molecular modeling and simulations to investigate, with atomistic details, the isoform dependence of actin-myosin interactions in the rigor state. By combining electron microscopy-based docking with homology modeling, we have constructed three all-atom models for human cardiac α and β and rabbit fast skeletal muscle myosin in complex with three actin subunits in the rigor state. Starting from these models, we have performed extensive all-atom molecular dynamics (MD) simulations (total of 100 ns per system) and then used the MD trajectories to calculate actin-myosin binding free energies with contributions from both electrostatic and nonpolar forces. Our binding calculations are in good agreement with the experimental finding of isoform-dependent differences in actin binding affinity between these myosin isoforms. Such differences are traced to changes in actin-myosin electrostatic interactions (i.e., hydrogen bonds and salt bridges) that are highly dynamic and involve several flexible actin-binding loops. By partitioning the actin-myosin binding free energy to individual myosin residues, we have also identified key myosin residues involved in the actin-myosin interactions, some of which were previously validated experimentally or implicated in cardiomyopathy mutations, and the rest make promising targets for future mutational experiments. PMID:24224850

  14. Mesenchymal chemotaxis requires selective inactivation of Myosin II at the leading edge via a non-canonical PLCγ/PKCα pathway

    PubMed Central

    Asokan, Sreeja B.; Johnson, Heath E.; Rahman, Anisur; King, Samantha J.; Rotty, Jeremy D.; Lebedeva, Irina P.; Haugh, Jason M.; Bear, James E.

    2014-01-01

    Summary Chemotaxis, migration towards soluble chemical cues, is critical for processes such as wound healing and immune surveillance, and is exhibited by various cell types from rapidly-migrating leukocytes to slow-moving mesenchymal cells. To interrogate the mechanisms involved in mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of the chemoattractant PDGF. Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mTOR signaling, are dispensable for chemotaxis to PDGF. Instead, we find that local inactivation of Myosin IIA, through a non-canonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of TIRF imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge is required for mesenchymal chemotaxis. PMID:25482883

  15. Rho GTPases and the Downstream Effectors Actin-related Protein 2/3 (Arp2/3) Complex and Myosin II Induce Membrane Fusion at Self-contacts*

    PubMed Central

    Sumida, Grant M.; Yamada, Soichiro

    2015-01-01

    Actin regulation is required for membrane activities that drive cell adhesion and migration. The Rho GTPase family plays critical roles in actin and membrane dynamics; however, the roles of the Rho GTPase family are not limited to cell adhesion and migration. Using micron-sized obstacles to induce the formation of self-contacts in epithelial cells, we previously showed that self-adhesion is distinct from cell-to-cell adhesion in that self-contacts are eliminated by membrane fusion. In the current study, we identified Rho GTPases, RhoA, Rac1, and Cdc42, as potential upstream regulators of membrane fusion. The RhoA downstream effector myosin II is required for fusion as the expression of mutant myosin light chain reduced membrane fusion. Furthermore, an inhibitor of the Arp2/3 complex, a downstream effector of Rac1 and Cdc42, also reduced self-contact-induced membrane fusion. At self-contacts, while the concentration of E-cadherin diminished, the intensity of GFP-tagged Arp3 rapidly fluctuated then decreased and stabilized after membrane fusion. Taken together, these data suggest that the Arp2/3 complex-mediated actin polymerization brings two opposing membranes into close apposition by possibly excluding E-cadherin from contact sites, thus promoting membrane fusion at self-contacts. PMID:25527498

  16. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    PubMed Central

    Reddy, Anireddy SN; Day, Irene S

    2001-01-01

    Background Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. Results Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. Conclusions Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined. PMID:11516337

  17. Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles*

    PubMed Central

    Nagy, Stanislav; Rock, Ronald S.

    2010-01-01

    Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses. PMID:20538587

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

    PubMed

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

    2016-03-29

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

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

    PubMed Central

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

    2016-01-01

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T (0.8–0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s−1 at high load to ∼8 nm⋅hs−1 and 6,000 s−1 at low load. Increases in sarcomere length (1.9–2.2 μm) and external [Ca2+]o (1–2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca2+-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

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

    PubMed

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

    2014-06-15

    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 simulations 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

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

    PubMed Central

    Santiago, Ednalise; Akamine, Pearl; Snider, Jamie; Wong, Victoria; Jessulat, Matthew; Deineko, Viktor; Gagarinova, Alla; Aoki, Hiroyuki; Minic, Zoran; Phanse, Sadhna; San Antonio, Andrea; Cubano, Luis A.; Rymond, Brian C.; Babu, Mohan; Stagljar, Igor; Rodriguez-Medina, Jose R.

    2016-01-01

    Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis. PMID:26921299

  2. Opaque1 Encodes a Myosin XI Motor Protein That Is Required for Endoplasmic Reticulum Motility and Protein Body Formation in Maize Endosperm[C][W][OA

    PubMed Central

    Wang, Guifeng; Wang, Fang; Wang, Gang; Wang, Fei; Zhang, Xiaowei; Zhong, Mingyu; Zhang, Jin; Lin, Dianbin; Tang, Yuanping; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70–interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture. PMID:22892319

  3. Stochastic dynamics of small ensembles of non-processive molecular motors: The parallel cluster model

    NASA Astrophysics Data System (ADS)

    Erdmann, Thorsten; Albert, Philipp J.; Schwarz, Ulrich S.

    2013-11-01

    Non-processive molecular motors have to work together in ensembles in order to generate appreciable levels of force or movement. In skeletal muscle, for example, hundreds of myosin II molecules cooperate in thick filaments. In non-muscle cells, by contrast, small groups with few tens of non-muscle myosin II motors contribute to essential cellular processes such as transport, shape changes, or mechanosensing. Here we introduce a detailed and analytically tractable model for this important situation. Using a three-state crossbridge model for the myosin II motor cycle and exploiting the assumptions of fast power stroke kinetics and equal load sharing between motors in equivalent states, we reduce the stochastic reaction network to a one-step master equation for the binding and unbinding dynamics (parallel cluster model) and derive the rules for ensemble movement. We find that for constant external load, ensemble dynamics is strongly shaped by the catch bond character of myosin II, which leads to an increase of the fraction of bound motors under load and thus to firm attachment even for small ensembles. This adaptation to load results in a concave force-velocity relation described by a Hill relation. For external load provided by a linear spring, myosin II ensembles dynamically adjust themselves towards an isometric state with constant average position and load. The dynamics of the ensembles is now determined mainly by the distribution of motors over the different kinds of bound states. For increasing stiffness of the external spring, there is a sharp transition beyond which myosin II can no longer perform the power stroke. Slow unbinding from the pre-power-stroke state protects the ensembles against detachment.

  4. Stochastic dynamics of small ensembles of non-processive molecular motors: The parallel cluster model

    SciTech Connect

    Erdmann, Thorsten; Albert, Philipp J.; Schwarz, Ulrich S.

    2013-11-07

    Non-processive molecular motors have to work together in ensembles in order to generate appreciable levels of force or movement. In skeletal muscle, for example, hundreds of myosin II molecules cooperate in thick filaments. In non-muscle cells, by contrast, small groups with few tens of non-muscle myosin II motors contribute to essential cellular processes such as transport, shape changes, or mechanosensing. Here we introduce a detailed and analytically tractable model for this important situation. Using a three-state crossbridge model for the myosin II motor cycle and exploiting the assumptions of fast power stroke kinetics and equal load sharing between motors in equivalent states, we reduce the stochastic reaction network to a one-step master equation for the binding and unbinding dynamics (parallel cluster model) and derive the rules for ensemble movement. We find that for constant external load, ensemble dynamics is strongly shaped by the catch bond character of myosin II, which leads to an increase of the fraction of bound motors under load and thus to firm attachment even for small ensembles. This adaptation to load results in a concave force-velocity relation described by a Hill relation. For external load provided by a linear spring, myosin II ensembles dynamically adjust themselves towards an isometric state with constant average position and load. The dynamics of the ensembles is now determined mainly by the distribution of motors over the different kinds of bound states. For increasing stiffness of the external spring, there is a sharp transition beyond which myosin II can no longer perform the power stroke. Slow unbinding from the pre-power-stroke state protects the ensembles against detachment.

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

    PubMed

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

    2002-01-01

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

  6. Mechanobiology of bone marrow stem cells: from myosin-II forces to compliance of matrix and nucleus

    PubMed Central

    Shin, Jae-Won; Swift, Joe; Ivanovska, Irena; Spinler, Kyle R.; Buxboim, Amnon; Discher, Dennis E.

    2014-01-01

    Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly effect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells. PMID:23790394

  7. p114RhoGEF governs cell motility and lumen formation during tubulogenesis through a ROCK-myosin-II pathway.

    PubMed

    Kim, Minji; M Shewan, Annette; Ewald, Andrew J; Werb, Zena; Mostov, Keith E

    2015-12-01

    Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis.

  8. Myosin flexibility: structural domains and collective vibrations.

    PubMed

    Navizet, Isabelle; Lavery, Richard; Jernigan, Robert L

    2004-02-15

    The movement of the myosin motor along an actin filament involves a directed conformational change within the cross-bridge formed between the protein and the filament. Despite the structural data that has been obtained on this system, little is known of the mechanics of this conformational change. We have used existing crystallographic structures of three conformations of the myosin head, containing the motor domain and the lever arm, for structural comparisons and mechanical studies with a coarse-grained elastic network model. The results enable us to define structurally conserved domains within the protein and to better understand myosin flexibility. Notably they point to the role of the light chains in rigidifying the lever arm and to changes in flexibility as a consequence of nucleotide binding.

  9. The role of myosin 1c and myosin 1b in surfactant exocytosis

    PubMed Central

    Kittelberger, Nadine; Breunig, Markus; Martin, René; Knölker, Hans-Joachim; Miklavc, Pika

    2016-01-01

    ABSTRACT Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression. PMID:26940917

  10. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    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. PMID:23923011

  11. Myosin-V stepping kinetics: a molecular model for processivity.

    PubMed

    Rief, M; Rock, R S; Mehta, A D; Mooseker, M S; Cheney, R E; Spudich, J A

    2000-08-15

    Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

  12. Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro

    NASA Technical Reports Server (NTRS)

    Conrad, A. H.; Jaffredo, T.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

  13. The fungal type II myosin in Penicillium marneffei, MyoB, is essential for chitin deposition at nascent septation sites but not actin localization.

    PubMed

    Cánovas, David; Boyce, Kylie J; Andrianopoulos, Alex

    2011-03-01

    Cytokinesis is essential for proliferative growth but also plays equally important roles during morphogenesis and development. The human pathogen Penicillium marneffei is capable of dimorphic switching in response to temperature, growing in a multicellular filamentous hyphal form at 25°C and in a unicellular yeast form at 37°C. P. marneffei also undergoes asexual development at 25°C to produce multicellular differentiated conidiophores. Thus, P. marneffei exhibits cell division with and without cytokinesis and division by budding and fission, depending on the cell type. The type II myosin gene, myoB, from P. marneffei plays important roles in the morphogenesis of these cell types. Deletion of myoB leads to chitin deposition defects at sites of cell division without perturbing actin localization. In addition to aberrant hyphal cells, distinct conidiophore cell types are lacking due to malformed septa and nuclear division defects. At 37°C, deletion of myoB prevents uninucleate yeast cell formation, instead producing long filaments resembling hyphae at 25°C. The ΔmyoB cells also often lyse due to defects in cell wall biogenesis. Thus, MyoB is essential for correct morphogenesis of all cell types regardless of division mode (budding or fission) and defines differences between the different types of growth. PMID:21131434

  14. Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation

    PubMed Central

    Ye, Qilu; Yang, Yidai; van Staalduinen, Laura; Crawley, Scott William; Liu, Linda; Brennan, Stephanie; Côté, Graham P.; Jia, Zongchao

    2016-01-01

    The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain. PMID:27211275

  15. Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

    PubMed Central

    Kaiser, Christian M.; Bujalowski, Paul J.; Ma, Liang; Anderson, John; Epstein, Henry F.; Oberhauser, Andres F.

    2012-01-01

    Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail. PMID:22824286

  16. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  17. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments.

  18. Motor Training Promotes Both Synaptic and Intrinsic Plasticity of Layer II/III Pyramidal Neurons in the Primary Motor Cortex

    PubMed Central

    Kida, Hiroyuki; Tsuda, Yasumasa; Ito, Nana; Yamamoto, Yui; Owada, Yuji; Kamiya, Yoshinori; Mitsushima, Dai

    2016-01-01

    Motor skill training induces structural plasticity at dendritic spines in the primary motor cortex (M1). To further analyze both synaptic and intrinsic plasticity in the layer II/III area of M1, we subjected rats to a rotor rod test and then prepared acute brain slices. Motor skill consistently improved within 2 days of training. Voltage clamp analysis showed significantly higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate (AMPA/NMDA) ratios and miniature EPSC amplitudes in 1-day trained rats compared with untrained rats, suggesting increased postsynaptic AMPA receptors in the early phase of motor learning. Compared with untrained controls, 2-days trained rats showed significantly higher miniature EPSC amplitude and frequency. Paired-pulse analysis further demonstrated lower rates in 2-days trained rats, suggesting increased presynaptic glutamate release during the late phase of learning. One-day trained rats showed decreased miniature IPSC frequency and increased paired-pulse analysis of evoked IPSC, suggesting a transient decrease in presynaptic γ-aminobutyric acid (GABA) release. Moreover, current clamp analysis revealed lower resting membrane potential, higher spike threshold, and deeper afterhyperpolarization in 1-day trained rats—while 2-days trained rats showed higher membrane potential, suggesting dynamic changes in intrinsic properties. Our present results indicate dynamic changes in glutamatergic, GABAergic, and intrinsic plasticity in M1 layer II/III neurons after the motor training. PMID:27193420

  19. Heartbeat control in leeches. II. Fictive motor pattern.

    PubMed

    Wenning, Angela; Hill, Andrew A V; Calabrese, Ronald L

    2004-01-01

    The rhythmic beating of the tube-like hearts in the medicinal leech is driven and coordinated by rhythmic activity in segmental heart motor neurons. The motor neurons are controlled by rhythmic inhibitory input from a network of heart interneurons that compose the heartbeat central pattern generator. In the preceding paper, we described the constriction pattern of the hearts in quiescent intact animals and showed that one heart constricts in a rear-to-front wave (peristaltic coordination mode), while the other heart constricts in near unison over its length (synchronous coordination mode) and that they regularly switch coordination modes. Here we analyze intersegmental and side-to-side-coordination of the fictive motor pattern for heartbeat in denervated nerve cords. We show that the intersegmental phase relations among heart motor neurons in both coordination modes are independent of heartbeat period. This finding enables us to combine data from different experiments to form a detailed analysis of the relative phases, duty cycle, and intraburst spike frequency of the bursts of the segmental heart motor neurons. The fictive motor pattern and the constriction pattern seen in intact leeches closely match in their intersegmental and side-to-side coordination, indicating that sensory feedback is not necessary for properly phased intersegmental coordination. Moreover, the regular switches in coordination mode of the fictive motor pattern mimic those seen in intact animals indicating that these switches likely arise by a central mechanism.

  20. Structural insight into the UNC-45-myosin complex.

    PubMed

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

    2013-07-01

    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 in silico methods. Initially, the human UNC-45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B-MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosin-actin 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. PMID:23408646

  1. Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

    PubMed Central

    Johnson, Terezina M.; Rajfur, Zenon; Jacobson, Ken

    2007-01-01

    The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes. PMID:17538016

  2. On the kinetics that moves Myosin V

    NASA Astrophysics Data System (ADS)

    Maes, Christian; O'Kelly de Galway, Winny

    2015-10-01

    Molecular motor proteins such as Myosin V, Dynein or Kinesin are no ratchets, at least not with a flashing asymmetric potential; the crucial asymmetry is in the dynamical activity. We make that explicit in terms of a simple Markov model, emphasizing the kinetic (and non-thermodynamic) aspects of stochastic transport. The analysis shows the presence of a fluctuation symmetry in that part of the dynamical activity which is antisymmetric under reversal of trailing and leading head of the motor. The direction of the motor motion is determined by it.

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

    NASA Astrophysics Data System (ADS)

    Veksler, Alexander; Vavylonis, Dimitrios

    2010-03-01

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

  4. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

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

    PubMed Central

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

    2016-01-01

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

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

    PubMed

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

    2015-04-01

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

  7. A simple theory of motor protein kinetics and energetics. II.

    PubMed

    Qian, H

    2000-01-10

    A three-state stochastic model of motor protein [Qian, Biophys. Chem. 67 (1997) pp. 263-267] is further developed to illustrate the relationship between the external load on an individual motor protein in aqueous solution with various ATP concentrations and its steady-state velocity. A wide variety of dynamic motor behavior are obtained from this simple model. For the particular case of free-load translocation being the most unfavorable step within the hydrolysis cycle, the load-velocity curve is quasi-linear, V/Vmax = (cF/Fmax-c)/(1-c), in contrast to the hyperbolic relationship proposed by A.V. Hill for macroscopic muscle. Significant deviation from the linearity is expected when the velocity is less than 10% of its maximal (free-load) value--a situation under which the processivity of motor diminishes and experimental observations are less certain. We then investigate the dependence of load-velocity curve on ATP (ADP) concentration. It is shown that the free load Vmax exhibits a Michaelis-Menten like behavior, and the isometric Fmax increases linearly with ln([ATP]/[ADP]). However, the quasi-linear region is independent of the ATP concentration, yielding an apparently ATP-independent maximal force below the true isometric force. Finally, the heat production as a function of ATP concentration and external load are calculated. In simple terms and solved with elementary algebra, the present model provides an integrated picture of biochemical kinetics and mechanical energetics of motor proteins. PMID:10631478

  8. Mechanical coupling in myosin V: a simulation study.

    PubMed

    Ovchinnikov, Victor; Trout, Bernhardt L; Karplus, Martin

    2010-01-29

    Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The interdomain coupling in myosin V is studied with restrained targeted molecular dynamics using an all-atom representation in explicit solvent. To elucidate the origin of the conformational change due to the binding of ATP, targeting forces are applied to small sets of atoms (the forcing sets, FSs) in the direction of their displacement from the rigor conformation, which has a closed actin-binding cleft, to the post-rigor conformation, in which the cleft is open. The "minimal" FS that results in extensive structural changes in the overall myosin conformation is composed of ATP, switch 1, and the nearby HF, HG, and HH helices. Addition of switch 2 to the FS is required to achieve a complete opening of the actin-binding cleft. The restrained targeted molecular dynamics simulations reveal the mechanical coupling pathways between (i) the nucleotide-binding pocket (NBP) and the actin-binding cleft, (ii) the NBP and the converter, and (iii) the actin-binding cleft and the converter. Closing of the NBP due to ATP binding is tightly coupled to the opening of the cleft and leads to the rupture of a key hydrogen bond (F441N/A684O) between switch 2 and the SH1 helix. The actin-binding cleft may mediate the rupture of this bond via a connection between the HW helix, the relay helix, and switch 2. The findings are consistent with experimental studies and a recent normal mode analysis. The present method is expected to be useful more generally in studies of interdomain coupling in proteins.

  9. Tuning myosin-driven sorting on cellular actin networks

    PubMed Central

    Hariadi, Rizal F; Sommese, Ruth F; Sivaramakrishnan, Sivaraj

    2015-01-01

    Myosin V and VI are antagonistic motors that cohabit membrane vesicles in cells. A systematic study of their collective function, however, is lacking and forms the focus of this study. We functionally reconstitute a two-dimensional actin-myosin interface using myosin V and VI precisely patterned on DNA nanostructures, in combination with a model keratocyte actin meshwork. While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm. This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold. Pairing computation with experimental observations suggests that the ratio of motor stall forces is a key determinant of the observed competitive outcomes. Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type. DOI: http://dx.doi.org/10.7554/eLife.05472.001 PMID:25738229

  10. Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor.

    PubMed

    Takano, Mitsunori; Terada, Tomoki P; Sasai, Masaki

    2010-04-27

    The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor.

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

    PubMed

    Ohyama, A; Komiya, Y; Igarashi, M

    2001-02-01

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

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

    PubMed

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

    2015-03-30

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

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

    PubMed

    Geeves, Michael A

    2016-08-01

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

  14. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

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

    PubMed

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

    2016-01-18

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

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

    PubMed Central

    Pollock, Lana M.; Chou, Shih-Wei

    2016-01-01

    The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing. PMID:26754648

  17. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-07-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo.

  18. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  19. Functional roles for myosin 1c in cellular signaling pathways

    PubMed Central

    Bond, Lisa M.; Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Cellular signaling pathways underlie the transfer of information throughout the cell and to adjoining cells and so govern most critical cellular functions. Increasing evidence points to the molecular motor myosin 1c as a prominent player in many signaling cascades, from the integrin-dependent signaling involved in cell migration to the signaling events underlying insulin resistance. Myosin 1c functions on these pathways both via an important role in regulating lipid raft recycling and also via direct involvement in signaling cascades. This review provides an overview of the functional involvement of myosin 1c in cellular signaling and discusses the possible potential for myosin 1c as a target for drug-based treatments for human diseases. PMID:23022959

  20. Still and rotating myosin clusters determine cytokinetic ring constriction

    PubMed Central

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  1. Reversible movement of switch 1 loop of myosin determines actin interaction

    PubMed Central

    Kintses, Bálint; Gyimesi, Máté; Pearson, David S; Geeves, Michael A; Zeng, Wei; Bagshaw, Clive R; Málnási-Csizmadia, András

    2007-01-01

    The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin–myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general. PMID:17213877

  2. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  3. Plasmodium falciparum myosins: transcription and translation during asexual parasite development.

    PubMed

    Chaparro-Olaya, Jacqueline; Margos, Gabriele; Coles, Deborah J; Dluzewski, Anton R; Mitchell, Graham H; Wasserman, Moisés M; Pinder, Jennifer C

    2005-04-01

    Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically; accordingly, we refer to the two latest additions as Pfmyo-E and Pfmyo-F. Both new myosins contain regions characteristic of the functional motor domain of "true" myosins and, unusually for P. falciparum myosins, Pfmyo-F encodes two consensus IQ light chain-binding motifs. Phylogenetic analysis of the 17 currently known apicomplexan myosins together with one representative of each myosin class clusters all but one of the apicomplexan sequences together in Class XIV. This refines the earlier definition of the Class XIV Subclasses XIVa and XIVb. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Thus: Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation. It is known that Pfmyo-A and Pfmyo-B are synthesised during late schizogony and we now show that Pfmyo-D expression is also temporally regulated to late trophozoites and schizonts where it distributes close to segregating nuclei. Thus, in asexual stages myosin synthesis does not always parallel transcript accumulation, showing that translation is also regulated. The implication is that the mRNAs are either subjected to turnover, synthesised and degraded, or that they are sequestered in an inactivate form until required for protein synthesis.

  4. Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth.

    PubMed

    Takeshita, Norio; Yamashita, Shuichi; Ohta, Akinori; Horiuchi, Hiroyuki

    2006-03-01

    The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.

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

    PubMed

    Silverman-Gavrila, Rosalind V; Forer, Arthur

    2003-06-01

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

  6. Leveraging the membrane-cytoskeleton interface with myosin-1

    PubMed Central

    McConnell, Russell E.; Tyska, Matthew J.

    2010-01-01

    Class 1 myosins are small motor proteins with the ability to simultaneously bind to actin filaments and cellular membranes. Given their ability to generate mechanical force, and their high prevalence in many cell types, these molecules are well positioned to carry out a number of important biological functions at the interface of membrane and the actin cytoskeleton. Indeed, recent studies implicate these motors in endocytosis, exocytosis, release of extracellular vesicles, and the regulation of tension between membrane and the cytoskeleton. Many class 1 myosins also exhibit a load-dependent mechano-chemical cycle that enables them to maintain tension for long periods of time without hydrolyzing ATP. These properties put myosins-1 in a unique position to regulate dynamic membrane-cytoskeleton interactions and respond to physical forces during these events. PMID:20471271

  7. Thirteen is enough: the myosins of Dictyostelium discoideum and their light chains

    PubMed Central

    Kollmar, Martin

    2006-01-01

    Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree

  8. Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

    PubMed

    Sari-Hassoun, Meryem; Clement, Marie-Jeanne; Hamdi, Imane; Bollot, Guillaume; Bauvais, Cyril; Joshi, Vandana; Toma, Flavio; Burgo, Andrea; Cailleret, Michel; Rosales-Hernández, Martha Cecilia; Macias Pérez, Martha Edith; Chabane-Sari, Daoudi; Curmi, Patrick A

    2016-02-15

    Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research. PMID:26707799

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

    NASA Astrophysics Data System (ADS)

    Jack, Amanda; Lowe, Ian; Tehver, Riina

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

  10. Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

    PubMed Central

    Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, John; Van Engelenburg, Schuyler B.; Ohi, Ryoma; Burnette, Dylan T.

    2016-01-01

    Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled. PMID:26960797

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

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  12. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

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

    PubMed

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

    2016-06-21

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

  14. Engineering myosins for long-range transport on actin filaments.

    PubMed

    Schindler, Tony D; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1).

  15. Engineering myosins for long-range transport on actin filaments

    NASA Astrophysics Data System (ADS)

    Schindler, Tony D.; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s-1.

  16. Engineering myosins for long-range transport on actin filaments.

    PubMed

    Schindler, Tony D; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1). PMID:24240432

  17. Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.

    PubMed

    Clow, P A; Chen, T; Chisholm, R L; McNally, J G

    2000-06-01

    During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

  18. A role for Fas II in the stabilization of motor neuron branches during pruning in Drosophila.

    PubMed

    Hebbar, Sarita; Fernandes, Joyce J

    2005-09-01

    During insect metamorphosis, the nervous system is extensively remodeled resulting in the development of new circuits that will execute adult-specific behaviors. The peripheral remodeling seen during development of innervation to the Dorsal Longitudinal (flight) Muscle (DLM) in Drosophila involves an initial retraction of larval neuromuscular junctions followed by adult-specific branch outgrowth. Subsequently, a phase of pruning occurs during which motor neuron branches are pruned back to reveal the stereotypic pattern of multiple contact points (or arbors) along the length of each DLM fiber. In this study, we show that the cell adhesion molecule, Fasciclin II (Fas II), is important for generating the stereotypic pattern. In Fas II hypomorphs, the number of contact points is increased, and the phenotype is rescued by targeted expression of Fas II in either synaptic partner. Arbor development has three distinct phases: outgrowth and elaboration, pruning and stabilization, and expansion of stabilized arbors. Fas II is expressed during the first two phases. A subset of branches is labeled during the elaboration phase, which is likely to initiate a stabilization pathway allowing branches to survive the pruning phase. However, since not all Fas II positive branches are retained, we propose that it primes branches for stabilization. Our data suggest that Fas II functions to restrict branch length and arbor expanse.

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

    PubMed

    Soldati, Thierry

    2003-06-01

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

  20. Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

    PubMed Central

    Chen, Tiane; Hubbard, Ann; Murtazina, Rakhilya; Price, Jennifer; Yang, Jianbo; Cha, Boyoung; Sarker, Rafiquel; Donowitz, Mark

    2014-01-01

    ABSTRACT The intestinal brush border Na+/H+ exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus. PMID:24928903

  1. Analysis of the interactions between Rab GTPases and class V myosins.

    PubMed

    Lindsay, Andrew J; Miserey-Lenkei, Stéphanie; Goud, Bruno

    2015-01-01

    Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli's Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid "living chip" assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation. PMID:25800833

  2. A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

    PubMed Central

    Dror, Amiel A.; Song, Lin; Ron, Uri; Tan, Joshua T.; Shitrit, Alina Starovolsky; Fuchs, Helmut; Hasson, Tama; Ben-Tal, Nir; Sweeney, H. Lee; de Angelis, Martin Hrabe; Steel, Karen P.; Avraham, Karen B.

    2008-01-01

    Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness. PMID:18833301

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

    SciTech Connect

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

    2008-04-25

    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.

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

    PubMed Central

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

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

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

    PubMed

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

    2016-10-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  6. Molecular motors: nature's nanomachines.

    PubMed

    Tyreman, M J A; Molloy, J E

    2003-12-01

    Molecular motors are protein-based machines that convert chemical potential energy into mechanical work. This paper aims to introduce the non-specialist reader to molecular motors, in particular, acto-myosin, the prototype system for motor protein studies. These motors produce their driving force from changes in chemical potential arising directly from chemical reactions and are responsible for muscle contraction and a variety of other cell motilities.

  7. Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V

    PubMed Central

    Rogers, Stephen L.; Karcher, Ryan L.; Roland, Joseph T.; Minin, Alexander A.; Steffen, Walter; Gelfand, Vladimir I.

    1999-01-01

    Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161–164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis. PMID:10491390

  8. An invertebrate smooth muscle with striated muscle myosin filaments

    PubMed Central

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-01-01

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components. PMID:26443857

  9. Mechanobiology of bone marrow stem cells: from myosin-II forces to compliance of matrix and nucleus in cell forms and fates.

    PubMed

    Shin, Jae-Won; Swift, Joe; Ivanovska, Irena; Spinler, Kyle R; Buxboim, Amnon; Discher, Dennis E

    2013-10-01

    Adult stem cells and progenitors are of great interest for their clinical application as well as their potential to reveal deep sensitivities to microenvironmental factors. The bone marrow is a niche for at least two types of stem cells, and the prototype is the hematopoietic stem cell/progenitors (HSC/Ps), which have saved many thousands of patients for several decades now. In bone marrow, HSC/Ps interact functionally with marrow stromal cells that are often referred to as mesenchymal stem cells (MSCs) or derivatives thereof. Myosin and matrix elasticity greatly affect MSC function, and these mechanobiological factors are now being explored with HSC/Ps both in vitro and in vivo. Also emerging is a role for the nucleus as a mechanically sensitive organelle that is semi-permeable to transcription factors which are modified for nuclear entry by cytoplasmic mechanobiological pathways. Since therapies envisioned with induced pluripotent stem cells and embryonic stem cells generally involve in vitro commitment to an adult stem cell or progenitor, a very deep understanding of stem cell mechanobiology is essential to progress with these multi-potent cells.

  10. Structural Basis for Myosin V Discrimination Between Distinct Cargoes

    SciTech Connect

    Pashkova,N.; Jin, Y.; Ramaswamy, S.; Weisman, L.

    2006-01-01

    Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 {angstrom} of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180{sup o}. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.

  11. The association of myosin IB with actin waves in dictyostelium requires both the plasma membrane-binding site and actin-binding region in the myosin tail.

    PubMed

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

    2014-01-01

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

  12. Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport.

    PubMed

    Ali, M Yusuf; Kennedy, Guy G; Safer, Daniel; Trybus, Kathleen M; Sweeney, H Lee; Warshaw, David M

    2011-08-23

    Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.

  13. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  14. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system.

    PubMed

    Caldwell, James T; Melkani, Girish C; Huxford, Tom; Bernstein, Sanford I

    2012-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

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

    PubMed

    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.

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

    PubMed Central

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

    2011-01-01

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

  17. Mechanochemical tuning of myosin-I by the N-terminal region.

    PubMed

    Greenberg, Michael J; Lin, Tianming; Shuman, Henry; Ostap, E Michael

    2015-06-30

    Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post-power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms. PMID:26056287

  18. Regulation and function of the fission yeast myosins.

    PubMed

    East, Daniel A; Mulvihill, Daniel P

    2011-05-01

    It is now quarter of a century since the actin cytoskeleton was first described in the fission yeast, Schizosaccharomyces pombe. Since then, a substantial body of research has been undertaken on this tractable model organism, extending our knowledge of the organisation and function of the actomyosin cytoskeleton in fission yeast and eukaryotes in general. Yeast represents one of the simplest eukaryotic model systems that has been characterised to date, and its genome encodes genes for homologues of the majority of actin regulators and actin-binding proteins found in metazoan cells. The ease with which diverse methodologies can be used, together with the small number of myosins, makes fission yeast an attractive model system for actomyosin research and provides the opportunity to fully understand the biochemical and functional characteristics of all myosins within a single cell type. In this Commentary, we examine the differences between the five S. pombe myosins, and focus on how these reflect the diversity of their functions. We go on to examine the role that the actin cytoskeleton plays in regulating the myosin motor activity and function, and finally explore how research in this simple unicellular organism is providing insights into the substantial impacts these motors can have on development and viability in multicellular higher-order eukaryotes. PMID:21502135

  19. The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V-Actin Bond

    PubMed Central

    Gebhardt, J. Christof M.; Ökten, Zeynep; Rief, Matthias

    2010-01-01

    Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense. PMID:20338849

  20. Transport of ER vesicles on actin filaments in neurons by myosin V.

    PubMed

    Tabb, J S; Molyneaux, B J; Cohen, D L; Kuznetsov, S A; Langford, G M

    1998-11-01

    Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for

  1. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

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

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

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

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

    PubMed

    Jackson, Del R; Baker, Josh E

    2009-06-28

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

  4. Structure of the light chain-binding domain of myosin V

    PubMed Central

    Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C.; Grabarek, Zenon; Dominguez, Roberto

    2005-01-01

    Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM–IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein–protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. PMID:16120677

  5. Calcium can mobilize and activate myosin-VI

    PubMed Central

    Batters, Christopher; Brack, Dario; Ellrich, Heike; Averbeck, Beate; Veigel, Claudia

    2016-01-01

    The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux. PMID:26811464

  6. Redox-sensitive residue in the actin-binding interface of myosin.

    PubMed

    Moen, Rebecca J; Cornea, Sinziana; Oseid, Daniel E; Binder, Benjamin P; Klein, Jennifer C; Thomas, David D

    2014-10-24

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  7. Redox-sensitive residue in the actin-binding interface of myosin

    PubMed Central

    Moen, Rebecca J.; Cornea, Sinziana; Oseid, Daniel E.; Binder, Benjamin P.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  8. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

    PubMed

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-10-10

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

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

    PubMed

    Martin, Che L; Singh, Shaneen M

    2015-11-01

    Myosins (MYO) define a superfamily of motor proteins which facilitate movement along cytoskeletal actin filaments in an ATP-dependent manner. To date, over 30 classes of myosin have been defined that vary in their roles and distribution across different taxa. The multidomain tail of myosin is responsible for the observed functional differences in different myosin classes facilitating differential binding to different cargos. One domain found in this region, the FERM domain, is found in several diverse proteins and is involved in many biological functions ranging from cell adhesion and actin-driven cytoskeleton assembly to cell signaling. Recently, new classes of unconventional myosin have been identified in Tetrahymena thermophila. In this study, we have identified, modeled, and characterized eight FERM domains from the unconventional T. thermophila myosins as their complete functional MyTH4-FERM cassettes. Our results reveal notable sequence, structural, and electrostatic differences between T. thermophila and other characterized FERM domains. Specifically, T. thermophila FERM domains contain helical inserts or extensions, which contribute to significant differences in surface electrostatic profiles of T. thermophila myosin FERMs when compared to the conventional FERM domains. Analyses of the modeled domains reveal differences in key functional residues as well as phosphoinositide-binding signatures and affinities. The work presented here broadens the scope of our understanding of myosin classes and their inherent functions, and provides a platform for experimentalists to design rational experimental studies to test the functional roles for T. thermophila myosins. PMID:26492945

  10. Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed.

    PubMed

    Shaffer, Justin F; Kier, William M

    2012-01-15

    The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds.

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

    PubMed

    Martin, Che L; Singh, Shaneen M

    2015-11-01

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

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

    PubMed

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

    2003-01-01

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

  13. Force Generation by Membrane-Associated Myosin-I

    PubMed Central

    Pyrpassopoulos, Serapion; Arpağ, Göker; Feeser, Elizabeth A.; Shuman, Henry; Tüzel, Erkan; Ostap, E. Michael

    2016-01-01

    Vertebrate myosin-IC (Myo1c) is a type-1 myosin that links cell membranes to the cytoskeleton via its actin-binding motor domain and its phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding tail domain. While it is known that Myo1c bound to PtdIns(4,5)P2 in fluid-lipid bilayers can propel actin filaments in an unloaded motility assay, its ability to develop forces against external load on actin while bound to fluid bilayers has not been explored. Using optical tweezers, we measured the diffusion coefficient of single membrane-bound Myo1c molecules by force-relaxation experiments, and the ability of ensembles of membrane-bound Myo1c molecules to develop and sustain forces. To interpret our results, we developed a computational model that recapitulates the basic features of our experimental ensemble data and suggests that Myo1c ensembles can generate forces parallel to lipid bilayers, with larger forces achieved when the myosin works away from the plane of the membrane or when anchored to slowly diffusing regions. PMID:27156719

  14. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

    PubMed Central

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-01-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  16. Thiol reactivity as a sensor of rotation of the converter in myosin.

    PubMed

    Onishi, Hirofumi; Nitanai, Yasushi

    2008-04-25

    Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the "converter", which rotates by approximately 70 degrees upon the transition from the "nucleotide-free" state to the "pre-power stroke" state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, "SH1", was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.

  17. Metal cation controls myosin and actomyosin kinetics

    PubMed Central

    Tkachev, Yaroslav V; Ge, Jinghua; Negrashov, Igor V; Nesmelov, Yuri E

    2013-01-01

    We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time-resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number. PMID:24115140

  18. Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing.

    PubMed

    Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B

    2014-06-01

    Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

  19. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI.

    PubMed

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-14

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  20. Myosin: A Link between Streptococci and Heart

    NASA Astrophysics Data System (ADS)

    Krisher, Karen; Cunningham, Madeleine W.

    1985-01-01

    Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

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

    SciTech Connect

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

    1987-10-01

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

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

    SciTech Connect

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

    2013-05-01

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

  3. Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

    PubMed

    Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

    2015-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that

  4. The myosin inhibitor blebbistatin stabilizes the super-relaxed state in skeletal muscle.

    PubMed

    Wilson, Clyde; Naber, Nariman; Pate, Edward; Cooke, Roger

    2014-10-01

    The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX. PMID:25296316

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

    PubMed Central

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

    1995-01-01

    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

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

    PubMed

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

    1995-04-01

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

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

    PubMed

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

    2010-01-01

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

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

    PubMed

    Oishi, N; Adachi, H; Sutoh, K

    2000-05-26

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

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

    PubMed

    Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

    2011-12-01

    Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

  10. Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

    PubMed Central

    Noguchi, Tatsuhiko; Frank, Deborah J.; Isaji, Mamiko

    2009-01-01

    Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. PMID:19005209

  11. Functional analysis of the sensory motor pathway of resistance reflex in crayfish. II. Integration Of sensory inputs in motor neurons.

    PubMed

    Le Ray, D; Clarac, F; Cattaert, D

    1997-12-01

    The in vitro preparation of the fifth thoracic ganglion of the crayfish was used to analyze the connections supporting the monosynaptic reflex responses recorded from the depressor motor neurons (Dep MNs). Dep MNs are directly connected by the release-sensitive afferents from a proprioceptor, the coxo-basipodite chordotonal organ (CBCO), which is released by upward movements of the leg. Sine-wave movements, applied to the CBCO strand from the most released position, allowed us to stimulate the greatest part of release-sensitive CBCO fibers. Systematic intracellular recordings from all Dep MNs performed in high divalent cation saline allowed us to determine the connections between CBCO afferents and their postsynaptic Dep MNs: it highlighted the sequential activation of the different Dep MNs involved in the monosynaptic reflex. The convergence of different sensory afferents onto a given Dep MN, and the divergence of a given sensory afferent onto several Dep MNs illustrates the complexity of the sensory-motor reflex loops involved in the control of locomotion and posture. Electrophysiological experiments and simulations were performed to analyze the mechanisms by which Dep MNs integrate the large amount of sensory input that they receive. Paired intracellular recording experiments demonstrated that postsynaptic response shapes characteristic of both phasic and phaso-tonic afferents could be induced by varying the presynaptic firing frequency, whatever the postsynaptic Dep MN. Compartment model simulations were used to analyze the role of the sensory-motor synapse characteristics in the summation properties of postsynaptic MN. They demonstrated the importance of the postsynaptic compartment geometry, because large postsynaptic compartments allowed to generate greater excitatory postsynaptic potential (EPSP) summations than small ones. The results presented show that velocity information is the most effective to elicit large compound EPSPs in MNs. We therefore suggest

  12. High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label.

    PubMed

    Binder, Benjamin P; Cornea, Sinziana; Thompson, Andrew R; Moen, Rebecca J; Thomas, David D

    2015-06-30

    Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures. PMID:26056276

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

    SciTech Connect

    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.

    2010-10-22

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

  14. Myosin heavy chain 10 (MYH10) is required for centriole migration during the biogenesis of primary cilia.

    PubMed

    Hong, Hyowon; Kim, Jongshin; Kim, Joon

    2015-05-22

    The actin cytoskeleton has been implicated in the assembly of cilia, but roles of actin-dependent motor proteins in ciliogenesis remain unclear. Myosin heavy chain 10 (MYH10), one of the isoforms of non-muscle myosin II, is known to mediate centrosome reorientation during cell migration. Here we show that MYH10 is required for centriole migration to the apical plasma membrane, which occurs at the onset of ciliogenesis. Knockdown of MYH10 in RPE1 cells caused a reduction in the levels of cortical filamentous actin (F-actin) and its binding protein EZRIN. Moreover, both centriole migration and subsequent cilium assembly were defective in MYH10 depleted cells. We further found that MYH10 influences centrosomal recruitment of IFT88, which is required for the transport of building blocks to the ciliary tip. The role of MYH10 in IFT88 recruitment appears to be indirect in that there is a correlation between centriolar IFT88 levels and centriolar positions along the apical-basal axis during ciliogenesis. Our results indicate that MYH10 contributes to ciliogenesis in RPE1 cells by promoting cortical actin-dependent centriole migration.

  15. Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

    PubMed Central

    Root, D D; Reisler, E

    1992-01-01

    The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin. PMID:1420910

  16. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens.

    PubMed

    Yang, Long; Qin, Li; Liu, Guosheng; Peremyslov, Valera V; Dolja, Valerian V; Wei, Yangdou

    2014-09-23

    The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity. PMID:25201952

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

    PubMed

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

    1999-01-01

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

  18. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

    PubMed Central

    Yang, Long; Qin, Li; Liu, Guosheng; Peremyslov, Valera V.; Dolja, Valerian V.; Wei, Yangdou

    2014-01-01

    The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity. PMID:25201952

  19. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    PubMed Central

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-01

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor’s oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  20. Mapping interactions between myosin relay and converter domains that power muscle function.

    PubMed

    Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

    2014-05-01

    Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

  1. Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

    PubMed

    Bertrand, R; Capony, J P; Derancourt, J; Kassab, R

    1999-09-14

    We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could

  2. Interplay between crosslinkers and dynamic molecular motor-induced instabilities in the moderation of biopolymer organization

    NASA Astrophysics Data System (ADS)

    Smith, David; Humphrey, David; Ziebert, Falko; Zimmermann, Walter; Käs, Josef

    2006-03-01

    Structure and function of biological cells rely on the highly-dynamic self-organization of protein filaments to an intracellular cytoskeleton responsive to mechanical and chemical stimuli. While dissolving these complex cellular structures through Brownian motion is inherently slow (tens of minutes), changes in the activity of the molecular motor myosin II cause rapid order-disorder transitions within 1-2 minutes in reconstituted cytoskeletal actin networks. When motor-induced filament sliding decreases, actin network structure rapidly and reversibly self-organizes into various assemblies triggered by a nonlinear instability. Modulation of static crosslinker concentrations allow for a wide phase space of order ranging from nematics to compact asters & dense packing of motor-filament clusters. The observed isothermal transitions between disorder and self-organization illustrate that molecular motors can substantially contribute to dynamic cellular organization.

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

    PubMed

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

    2016-06-14

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

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

    PubMed Central

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

    2016-01-01

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

  5. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.

  6. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator. PMID:27273580

  7. Binding of chara Myosin globular tail domain to phospholipid vesicles.

    PubMed

    Nunokawa, Shun-Ya; Anan, Hiromi; Shimada, Kiyo; Hachikubo, You; Kashiyama, Taku; Ito, Kohji; Yamamoto, Keiichi

    2007-11-01

    Binding of Chara myosin globular tail domain to phospholipid vesicles was investigated quantitatively. It was found that the globular tail domain binds to vesicles made from acidic phospholipids but not to those made from neutral phospholipids. This binding was weakened at high KCl concentration, suggesting that the binding is electrostatic by nature. The dissociation constant for the binding of the globular tail domain to 20% phosphatidylserine vesicles (similar to endoplasmic reticulum in acidic phospholipid contents) at 150 mM KCl was 273 nM. The free energy change due to this binding calculated from the dissociation constant was -37.3 kJ mol(-1). Thus the bond between the globular tail domain and membrane phospholipids would not be broken when the motor domain of Chara myosin moves along the actin filament using the energy of ATP hydrolysis (DeltaG degrees ' = -30.5 kJ mol(-1)). Our results suggested that direct binding of Chara myosin to the endoplasmic reticulum membrane through the globular tail domain could work satisfactorily in Chara cytoplasmic streaming. We also suggest a possible regulatory mechanism of cytoplasmic streaming including phosphorylation-dependent dissociation of the globular tail domain from the endoplasmic reticulum membrane.

  8. Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.

    PubMed

    Katta, Santharam S; Sahasrabuddhe, Amogh A; Gupta, Chhitar M

    2009-04-01

    Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids. PMID:19121339

  9. Evaluation of Acanthamoeba myosin-IC as a potential therapeutic target.

    PubMed

    Martín-Navarro, Carmen M; Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E; Valladares, Basilio; Maciver, Sutherland K

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as "traction-mediated cytokinesis". We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

  10. Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

    PubMed Central

    Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E.; Valladares, Basilio; Maciver, Sutherland K.

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

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

    PubMed

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

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis. PMID:27548633

  12. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

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

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

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

  14. Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck?

    PubMed

    Ratti, Joyce; Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2011-04-01

    Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

  15. Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration

    PubMed Central

    Yumura, Shigehiko; Itoh, Go; Kikuta, Yumi; Kikuchi, Takeomi; Kitanishi-Yumura, Toshiko; Tsujioka, Masatsune

    2013-01-01

    Summary Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton. PMID:23430058

  16. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-01

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

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

    PubMed

    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.

  18. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

    The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures. PMID:27060364

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

    PubMed Central

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

    2014-01-01

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

  20. Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability.

    PubMed

    Lee, Chi F; Melkani, Girish C; Yu, Qin; Suggs, Jennifer A; Kronert, William A; Suzuki, Yoko; Hipolito, Lori; Price, Maureen G; Epstein, Henry F; Bernstein, Sanford I

    2011-03-01

    UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

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

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    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.

  2. Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

    PubMed

    Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

    2014-12-01

    The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean

  3. Conformational changes of myosin by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Lee, Ju.-Woon; Yook, Hong.-Sun; Lee, Kyong.-Haeng; Kim, Jae.-Hun; Kim, Woo.-Jung; Byun, Myung.-Woo

    2000-05-01

    Conformational and decompositional changes of bovine skeletal muscle myosin caused by gamma irradiation were studied for understanding the effects of irradiation treatment on myofibrillar proteins. Myosin solution and beef cuts were irradiated 0, 1, 3, 5 and 10 kGy. Competitive indirect enzyme linked immunosorbent assay (Ci-ELISA) showed that subunits of myosin were structurally modified with different patterns. Binding abilities of anti-myosin whole molecule and anti heavy meromyosin S-1 IgG, which were produced from rabbits, with irradiated myosin decreased in the same tendency depending upon the dose. Anti-light meromyosin IgG appeared to have the highest binding ability at 3 kGy. Irradiated beef cuts (≥5 kGy) could be identified by Ci-ELISA. Myosin solution became increasingly turbid with increasing dose. Hydrophobicity of myosin solution also increased by irradiation. Electrophoretic patterns showed that the myosin heavy chain disappeared and new bands were generated at higher molecular weight ranges.

  4. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

    PubMed

    Linari, Marco; Brunello, Elisabetta; Reconditi, Massimo; Fusi, Luca; Caremani, Marco; Narayanan, Theyencheri; Piazzesi, Gabriella; Lombardi, Vincenzo; Irving, Malcolm

    2015-12-10

    Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart. PMID:26560032

  5. Equilibrium muscle cross-bridge behavior. Theoretical considerations. II. Model describing the behavior of strongly-binding cross-bridges when both heads of myosin bind to the actin filament.

    PubMed Central

    Schoenberg, M

    1991-01-01

    A model has been developed for characterizing the interaction between strongly-binding myosin cross-bridges and actin in muscle fibers under equilibrium conditions where both heads of the myosin cross-bridge bind to actin. The model, that of Anderson and Schoenberg (1987. Biophys. J. 52:1077-1082) is quite similar to that of Schoenberg (1985. Biophys. J. 48:467-475), except that explicit account is taken of the fact that each crossbridge has two heads which can bind to actin. The key assumption that allows this model to explain a large body of data unexplained by the Schoenberg (1985) model is that the two crossbridge heads are not totally independent of one another after attachment. After the first head attaches, the second head is then free to attach only to an actin site distal to the first head. This means that when the more distally attached head subsequently detaches and reattaches (as the heads continually do), it will not reattach in a position of lesser strain and reduce the force it supports, but instead will remain attached in its strained position until the proximally attached head also detaches. This model gives an explanation for two important and otherwise unexplained observations made previously: it explains why at ionic strengths in the range of 50-120 mM, (a) the rate constant of force decay after a small stretch is a sigmoidal function of nucleotide analogue concentration, and (b) why in the presence of analogues or in rigor the rate constant of force decay after a small stretch is significantly slower than the rate constant for myosin subfragment-1 detachment from actin in solution. PMID:1932554

  6. Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum.

    PubMed

    Katta, Santharam S; Tammana, Trinadh V Satish; Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2010-06-15

    Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum. PMID:20501700

  7. Effect of nucleotides on the orientation and mobility of myosin subfragment-1 in ghost muscle fiber.

    PubMed

    Pronina, O E; Wrzosek, A; Dabrowska, R; Borovikov, Yu S

    2005-10-01

    Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.

  8. Polymer Nanocomposites as a Facile Method for Engineering Acto-Myosin Networks at the Interface

    NASA Astrophysics Data System (ADS)

    Caporizzo, Matthew; Sun, Yujie; Goldman, Yale; Composto, Russell; Nano-Bio Interface Center Collaboration

    2011-03-01

    Filamentous actin acts as the rails for the molecular motor myosin in muscle contraction and intercellular mass transport. Consequently, understanding the process by which actin organizes, polymerizes, and binds is fundamental for the design of myosin based actuators capable of responding to external stimuli. Starting with atomically smooth, freshly cleaved mica optically coupled to glass slides, a random copolymer nanoparticle composite is engineered for in situ single molecule TIRF/AFM studies with controlled roughness, electrostatic binding strength, and binding site density. Four distinct regimes of actin binding are observed; no attachment, end-on attachment, weak side-on attachment, and side-on immobilization. Transitions between regimes are likely to mark competition between the affinity to charged nanoparticles and the inherent resistance of the semi-rigid filaments to bending. Surface conditions optimal for actin immobilization are identified, and Myosin V stepping kinetics are studied on the artificially immobilized filaments, confirming filament support of motility. Supported by NSF grant DMR-0425780.

  9. Thin-disc piezoceramic ultrasonic motor. Part II: system construction and control.

    PubMed

    Yen, Chi Yung; Wen, Fuh Liang; Ouyang, Minsun

    2003-08-01

    Design and performance evaluation of an ultrasonic motor was discussed in [Wen et al., Thin-disc piezoelectric ultrasonic motor. Part I: design and performance evaluation, Ultrasonics]. Higher precision position control of piezoceramic ultrasonic motor depends on mechanical design and servo control of a very precise and adequate metrology. This paper proposes the design of a driving circuit and controller to deal with non-linearities behavior in the model of piezoceramic-driving ultrasonic motor. The performance of the driver and the effectiveness of the proposed controller are demonstrated by command inputs of sinusoidal and step signals. For comparison purpose, the ultrasonic motor is controlled using two methods: i.e., proportional-integral-derivative (PID) and sliding-mode control (SMC). It was proven that SMC would compensate automatically for unmodeled behaviors such as piezoceramic non-linearities and mechanical stick-slip phenomena. Furthermore, SMC scheme has been successfully applied to position tracking to demonstrate the excellent robust performance in noise rejection.

  10. Structural kinetics of myosin by transient time-resolved FRET

    PubMed Central

    Nesmelov, Yuri E.; Agafonov, Roman V.; Negrashov, Igor V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.

    2011-01-01

    For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. PMID:21245357

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

    PubMed

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

    2016-05-24

    Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution. PMID:27166421

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

    PubMed

    Masuda, Tadashi

    2015-06-01

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

  13. Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron

    PubMed Central

    Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

    2016-01-01

    Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons. PMID:27570482

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

    PubMed

    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.

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

    PubMed

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

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

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

    PubMed Central

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

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  17. Proper expression of myosin genes in transgenic nematodes.

    PubMed Central

    Fire, A; Waterston, R H

    1989-01-01

    Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product. Images PMID:2583105

  18. Visualization of the mechanochemical coupling in myosin V using deac-aminoATP

    NASA Astrophysics Data System (ADS)

    Sakaoto, Takeshi; Webb, Martin; Forgacs, Eva; White, Howard; Sellers, Jim

    2010-03-01

    MyosinV is a twoheaded motor, which moves processively along an actin with ADP-release as the rate limiting step. The kinetic cycles of the two heads are gated by the internal strain each places on the other Mechanical studies suggest that there is tight coupling (i.e., one ATP is hydrolyzed per power stroke). We investigated the coordination between the ATPase mechanism of the two heads-myosin Va and directly visualized the binding and dissociation of nucleotide molecules, while simultaneously observing the stepping motion of the myosin V as it moved along an actin filament. To do this, we used an fluorescent labeled ATP analog, deac-aminoATP, which shows a 20fold increase in fluorescent intensity when bound to the active site of myosinV I directly demonstrate tight coupling between myosin V movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision. Supported by K99/R00 NIH grant.

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

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2015-03-17

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

  1. Cross-reactivity of termite myosin; a potential allergen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin and myosin isoforms are common food allergens in crustaceans; such as, shrimp, lobster, and crab. Allergy to Shellfish is a prevalent and potentially long lasting disorder that can severely affect health and quality of life. Myosin and myosin isoforms of dust mites and cockroaches are simil...

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

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

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

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

    NASA Astrophysics Data System (ADS)

    Veksler, Alexander; Vavylonis, Dimitrios

    2011-03-01

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

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

    PubMed

    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.

  5. Nonmuscle Myosin IIA Regulates Intestinal Epithelial Barrier in vivo and Plays a Protective Role During Experimental Colitis.

    PubMed

    Naydenov, Nayden G; Feygin, Alex; Wang, Dongdong; Kuemmerle, John F; Harris, Gianni; Conti, Mary Anne; Adelstein, Robert S; Ivanov, Andrei I

    2016-01-01

    The actin cytoskeleton is a critical regulator of intestinal mucosal barrier permeability, and the integrity of epithelial adherens junctions (AJ) and tight junctions (TJ). Non muscle myosin II (NM II) is a key cytoskeletal motor that controls actin filament architecture and dynamics. While NM II has been implicated in the regulation of epithelial junctions in vitro, little is known about its roles in the intestinal mucosa in vivo. In this study, we generated a mouse model with an intestinal epithelial-specific knockout of NM IIA heavy chain (NM IIA cKO) and examined the structure and function of normal gut barrier, and the development of experimental colitis in these animals. Unchallenged NM IIA cKO mice showed increased intestinal permeability and altered expression/localization of several AJ/TJ proteins. They did not develop spontaneous colitis, but demonstrated signs of a low-scale mucosal inflammation manifested by prolapses, lymphoid aggregates, increased cytokine expression, and neutrophil infiltration in the gut. NM IIA cKO animals were characterized by a more severe disruption of the gut barrier and exaggerated mucosal injury during experimentally-induced colitis. Our study provides the first evidence that NM IIA plays important roles in establishing normal intestinal barrier, and protection from mucosal inflammation in vivo. PMID:27063635

  6. Nonmuscle Myosin IIA Regulates Intestinal Epithelial Barrier in vivo and Plays a Protective Role During Experimental Colitis

    PubMed Central

    Naydenov, Nayden G.; Feygin, Alex; Wang, Dongdong; Kuemmerle, John F.; Harris, Gianni; Conti, Mary Anne; Adelstein, Robert S.; Ivanov, Andrei I.

    2016-01-01

    The actin cytoskeleton is a critical regulator of intestinal mucosal barrier permeability, and the integrity of epithelial adherens junctions (AJ) and tight junctions (TJ). Non muscle myosin II (NM II) is a key cytoskeletal motor that controls actin filament architecture and dynamics. While NM II has been implicated in the regulation of epithelial junctions in vitro, little is known about its roles in the intestinal mucosa in vivo. In this study, we generated a mouse model with an intestinal epithelial-specific knockout of NM IIA heavy chain (NM IIA cKO) and examined the structure and function of normal gut barrier, and the development of experimental colitis in these animals. Unchallenged NM IIA cKO mice showed increased intestinal permeability and altered expression/localization of several AJ/TJ proteins. They did not develop spontaneous colitis, but demonstrated signs of a low-scale mucosal inflammation manifested by prolapses, lymphoid aggregates, increased cytokine expression, and neutrophil infiltration in the gut. NM IIA cKO animals were characterized by a more severe disruption of the gut barrier and exaggerated mucosal injury during experimentally-induced colitis. Our study provides the first evidence that NM IIA plays important roles in establishing normal intestinal barrier, and protection from mucosal inflammation in vivo. PMID:27063635

  7. Unconventional myosin ID is expressed in myelinating oligodendrocytes.

    PubMed

    Yamazaki, Reiji; Ishibashi, Tomoko; Baba, Hiroko; Yamaguchi, Yoshihide

    2014-10-01

    Myelin is a dynamic multilamellar structure that ensheathes axons and is crucial for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes that wrap many layers of plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we found that myosin ID (Myo1d), a class I myosin, is enriched in the rat CNS myelin fraction. Myo1d is an unconventional myosin and has been shown to be involved in membrane trafficking in the recycling pathway in an epithelial cell line. Western blotting revealed that Myo1d expression begins early in myelinogenesis and continues to increase into adulthood. The localization of Myo1d in CNS myelin has not been reported, and the function of Myo1d in vivo remains unknown. To demonstrate the expression of Myo1d in CNS myelin and to begin to explore the function of Myo1d in myelination, we produced a new antibody against Myo1d that has a high titer and specificity for rat Myo1d. By using this antibody, we demonstrated that Myo1d is expressed in rat CNS myelin and is especially abundant in abaxonal and adaxonal regions (the outer and inner cytoplasm-containing loops, respectively), but that expression is low in peripheral nervous system myelin. In culture, Myo1d was expressed in mature rat oligodendrocytes. Furthermore, an increase in expression of Myo1d during maturation of CNS white matter (cerebellum and corpus callosum) was demonstrated by histological analysis. These results suggest that Myo1d may be involved in the formation and/or maintenance of CNS myelin.

  8. Contractile properties, fiber types, and myosin isoforms in fast and slow muscles of hyperactive Japanese waltzing mice.

    PubMed

    Asmussen, Gerhard; Schmalbruch, Ina; Soukup, Tomás; Pette, Dirk

    2003-12-01

    This study focuses on the effects of neuromuscular hyperactivity on the contractile properties, fiber type composition, and myosin heavy chain (MHC) isoform expression of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles in Japanese waltzing mice (JWM) of the C57BL/6J-v2J strain. The same properties were studied in the homologous muscle of control CBA/J mice (CM). In comparison to CM, the JWM exhibited (i) longer activity periods, prolonged bouts of running and a higher food intake, (ii) slower twitch and tetanic contractions of both EDL and SOL muscles, decreased cold and post-tetanic potentiation of the EDL, as well as increased cold and post-tetanic depressions of the SOL. Electrophoretic analyses of MHC isoform revealed a shift toward slower isoforms in both EDL and SOL muscles of JWM as compared to the homologous muscles of CM, namely, a shift from the fastest MHCIIb to the MHCIId/x isoform in the EDL muscle and a shift from MHCIIa to MHCI in the SOL muscle. The latter also contained a higher percentage of type I fibers and displayed a higher capillary density than the SOL muscle of CM. These findings show that the inherently enhanced motor activity of the JWM leads to fiber type transitions in the direction of slower phenotypes. JWM thus represent a suitable model for studying fast-to-slow fiber transitions under the influence of spontaneous motor hyperactivity.

  9. Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

    PubMed Central

    Takagi, Yasuharu; Farrow, Rachel E.; Billington, Neil; Nagy, Attila; Batters, Christopher; Yang, Yi; Sellers, James R.; Molloy, Justin E.

    2014-01-01

    Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12–14 s−1) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor. PMID:24753602

  10. Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization.

    PubMed

    Yau, Richard G; Peng, Yutian; Valiathan, Rajeshwari R; Birkeland, Shanda R; Wilson, Thomas E; Weisman, Lois S

    2014-03-10

    Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2, binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism that detaches myosin V from select cargoes.

  11. Polarized myosin produces unequal-size daughters during asymmetric cell division.

    PubMed

    Ou, Guangshuo; Stuurman, Nico; D'Ambrosio, Michael; Vale, Ronald D

    2010-10-29

    Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.

  12. Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction.

    PubMed

    Burghardt, Thomas P; Josephson, Matthew P; Ajtai, Katalin

    2011-09-13

    Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.

  13. Dynamic exchange of myosin molecules between thick filaments.

    PubMed

    Saad, A D; Pardee, J D; Fischman, D A

    1986-12-01

    To examine thick filament assembly and myosin exchange, a fluorescence energy transfer assay has been established. Assembly-competent myosin molecules labeled with the sulfhydryl-specific fluorochromes 5-(2-[(iodoacetyl)-amino]ethyl)aminonaphthalene-1-sulfonic acids (IAEDANS) or 5-iodoacetamidofluorescein (IAF) were prepared. Using IAEDANS-labeled myosin as fluorescence donor and IAF-labeled myosin as acceptor, thick filament formation was followed by the decrease in donor fluorescence at 0.1 M KCl/10 mM potassium phosphate, pH 6.9. The critical concentration of myosin--i.e., that concentration that remained unassembled at equilibrium with fully formed filaments--was 40 nM. In FET and 125I-labeled myosin incorporation assays, extensive exchange of myosin between thick filaments was observed. The presence of a critical concentration and the measurements of extensive exchange suggest a dynamic equilibrium between fully polymerized myosin and a small pool of soluble myosin.

  14. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    PubMed

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro

    2016-06-01

    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  15. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  16. Mouse Nuclear Myosin I Knock-Out Shows Interchangeability and Redundancy of Myosin Isoforms in the Cell Nucleus

    PubMed Central

    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

    Background Nuclear myosin I (NM1) is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein (Myo1c). Located on the 11th 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. Methodology/Principal Findings 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. Conclusion/Significance 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. PMID:23593477

  17. Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1*

    PubMed Central

    Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2014-01-01

    Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (Vmax = 4 s−1), although their affinities for actin were high (Kactin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s−1, respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

  18. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    PubMed

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  19. Visible-Light-Driven Photoisomerization and Increased Rotation Speed of a Molecular Motor Acting as a Ligand in a Ruthenium(II) Complex.

    PubMed

    Wezenberg, Sander J; Chen, Kuang-Yen; Feringa, Ben L

    2015-09-21

    Toward the development of visible-light-driven molecular rotary motors, an overcrowded alkene-based ligand and the corresponding ruthenium(II) complex is presented. In our design, a 4,5-diazafluorenyl coordination motif is directly integrated into the motor function. The photochemical and thermal isomerization behavior has been studied by UV/Vis and NMR spectroscopy. Upon coordination to a Ru(II) bipyridine complex, the photoisomerization process can be driven by visible (λmax = 450 nm) instead of UV light and furthermore, a large increase of the speed of rotation is noted. DFT calculations point to a contraction of the diazafluorenyl lower half upon metal-coordination resulting in reduced steric hindrance in the "fjord region" of the molecule. Consequently, it is shown that metal-ligand interactions can play an important role in the adjustment of both photophysical and thermodynamic properties of molecular motors.

  20. Myosin Heavy Chain Composition of the Human Hyoglossus Muscle*

    PubMed Central

    Sokoloff, Alan J.; Daugherty, Megan; Li, Haiyan

    2013-01-01

    The human tongue muscle hyoglossus (HG) muscle is active in oro-motor behaviors encompassing a wide range of tongue movement speeds. Here we test the hypothesis that the human HG is composed of “uncommon” myosin heavy chain (MHC) isoforms MHCembryonic, MHCneonatal and MHCslow tonic as has been reported for other head and neck muscles active during kinematically diverse behaviors. Following reaction of human HG with antibodies specific for MHCI, MHCIIA, MHCII, MHCembryonic, MHCextraocular, MHCneonatal and MHCslow tonic only antibodies to MHCI, MHCIIA and MHCIIA-X label more than occasional muscle fibers. These antibodies describe five phenotypes with prevalence MHCIIA>MHCI>MHCI-IIX>MHCI-IIA>MHCIIX. In MHC composition, the human HG is thus similar to human appendicular muscles, the human tongue muscle styloglossus and many human head and neck muscles but different from human masseter and extraocular muscles which contain five or more MHC isoforms. PMID:19526266

  1. A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis elegans Motor Neurons

    PubMed Central

    Hoover, Christopher M.; Edwards, Stacey L.; Yu, Szi-chieh; Kittelmann, Maike; Richmond, Janet E.; Eimer, Stefan; Yorks, Rosalina M.; Miller, Kenneth G.

    2014-01-01

    Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II. PMID:24653209

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

    PubMed

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

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion. PMID:27498562

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

    PubMed

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

    2016-08-22

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

  4. Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis

    PubMed Central

    Vasquez, Claudia G.; Tworoger, Mike

    2014-01-01

    Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. Here, we show that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis. PMID:25092658

  5. Structural dynamics of the myosin relay helix by time-resolved EPR and FRET

    PubMed Central

    Agafonov, Roman V.; Negrashov, Igor V.; Tkachev, Yaroslav V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.; Nesmelov, Yuri E.

    2009-01-01

    We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeFx) or ADP.Pi (ADP.AlF4) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF4 was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition. PMID:19966224

  6. Chlamydia trachomatis inclusion membrane protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms

    PubMed Central

    Lutter, Erika I.; Barger, Alexandra C.; Nair, Vinod; Hackstadt, Ted

    2013-01-01

    Summary Chlamydia trachomatis replicates within a membrane bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion while the phosphorylated inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), and myosin IIA and IIB also colocalized with inactive MYPT1. The role of these proteins was examined in the context of host-cell exit mechanisms; cell lysis or extrusion of intact inclusions. Inhibition of myosin II or siRNA depletion of myosin IIA and IIB, MLC2, or MLCK reduced chlamydial extrusion thus favoring lytic events as the primary means of release. These studies provide insights into regulation of egress mechanisms by C. trachomatis. PMID:23727243

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

    SciTech Connect

    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.

    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.

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

    SciTech Connect

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

    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

  9. Cell and molecular biology of the fastest myosins.

    PubMed

    Higashi-Fujime, Sugie; Nakamura, Akio

    2009-01-01

    Chara myosin is a class XI plant myosin in green algae Chara corallina and responsible for fast cytoplasmic streaming. The Chara myosin exhibits the fastest sliding movement of F-actin at 60 mum/s as observed so far, 10-fold of the shortening speed of muscle. It has some distinct properties differing from those of muscle myosin. Although knowledge about Chara myosin is very limited at present, we have tried to elucidate functional bases of its characteristics by comparing with those of other myosins. In particular, we have built the putative atomic model of Chara myosin by using the homology-based modeling system and databases. Based on the putative structure of Chara myosin obtained, we have analyzed the relationship between structure and function of Chara myosin to understand its distinct properties from various aspects by referring to the accumulated knowledge on mechanochemical and structural properties of other classes of myosin, particularly animal and fungal myosin V. We will also discuss the functional significance of Chara myosin in a living cell.

  10. Modes and Mechanisms of T cell Motility: Roles for Confinement and Myosin-IIA

    PubMed Central

    Krummel, Matthew F.; Friedman, Rachel S.; Jacobelli, Jordan

    2014-01-01

    T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizes the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode of interaction with surfaces that is more conducive to forming a synapse with an antigen-presenting cell. PMID:24905977

  11. Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Sckolnick, Maria; Bookwalter, Carol S.; Hodges, Alex R.; Trybus, Kathleen M.; Lord, Matthew

    2014-01-01

    A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. PMID:24196839

  12. Myosin-10 and actin filaments are essential for mitotic spindle function

    PubMed Central

    Woolner, Sarah; O'Brien, Lori L.; Wiese, Christiane; Bement, William M.

    2008-01-01

    Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length. PMID:18606852

  13. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein

    PubMed Central

    1993-01-01

    In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium- density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells. PMID:8416982

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

    PubMed

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

    2005-10-14

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

  15. Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

    PubMed Central

    Lecuona, Emilia; Minin, Alexander; Trejo, Humberto E.; Chen, Jiwang; Comellas, Alejandro P.; Sun, Haiying; Grillo, Doris; Nekrasova, Oxana E.; Welch, Lynn C.; Szleifer, Igal; Gelfand, Vladimir I.; Sznajder, Jacob I.

    2009-01-01

    Summary Stimulation of Na+/K+-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na+/K+-ATPase recruitment to the plasma membrane by increasing the average speed of Na+/K+-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na+/K+-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na+/K+-ATPase-containing vesicles, as well as the Na+/K+-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na+/K+-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na+/K+-ATPase to the plasma membrane and thus increased activity. PMID:19808891

  16. Spatial memory deficits and motor coordination facilitation in cGMP-dependent protein kinase type II-deficient mice.

    PubMed

    Wincott, Charlotte M; Kim, Seonil; Titcombe, Roseann F; Tukey, David S; Girma, Hiwot K; Pick, Joseph E; Devito, Loren M; Hofmann, Franz; Hoeffer, Charles; Ziff, Edward B

    2013-01-01

    Activity-dependent trafficking of AMPA receptors to synapses regulates synaptic strength. Activation of the NMDA receptor induces several second messenger pathways that contribute to receptor trafficking-dependent plasticity, including the NO pathway, which elevates cGMP. In turn, cGMP activates the cGMP-dependent protein kinase type II (cGKII), which phosphorylates the AMPA receptor subunit GluA1 at serine 845, a critical step facilitating synaptic delivery in the mechanism of activity-dependent synaptic potentiation. Since cGKII is expressed in the striatum, amygdala, cerebral cortex, and hippocampus, it has been proposed that mice lacking cGKII may present phenotypic differences compared to their wild-type littermates in emotion-dependent tasks, learning and memory, and drug reward salience. Previous studies have shown that cGKII KO mice ingest higher amounts of ethanol as well as exhibit elevated anxiety levels compared to wild-type (WT) littermates. Here, we show that cGKII KO mice are significantly deficient in spatial learning while exhibiting facilitated motor coordination, demonstrating a clear dependence of memory-based tasks on cGKII. We also show diminished GluA1 phosphorylation in the postsynaptic density (PSD) of cGKII KO prefrontal cortex while in hippocampal PSD fractions, phosphorylation was not significantly altered. These data suggest that the role of cGKII may be more robust in particular brain regions, thereby impacting complex behaviors dependent on these regions differently.

  17. Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

    PubMed

    Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina; Chance, Mark R; Reisler, Emil

    2011-11-25

    Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function. PMID:21986200

  18. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    PubMed Central

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins. PMID:10827983

  19. Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments.

    PubMed

    Suzuki, Makoto; Kabir, Syed Rashel; Siddique, Md Shahjahan Parvez; Nazia, Umme Salma; Miyazaki, Takashi; Kodama, Takao

    2004-09-10

    Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.]. PMID:15313212

  20. Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

    PubMed Central

    Stauffer, Eric A.; Scarborough, John D.; Hirono, Moritoshi; Miller, Emilie D.; Shah, Kavita; Mercer, John A.; Holt, Jeffrey R.; Gillespie, Peter G.

    2009-01-01

    Summary In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells. PMID:16102537

  1. Preparation and Characterization of Myosin Proteins.

    ERIC Educational Resources Information Center

    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)

  2. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

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

    1986-03-01

    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/sup 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 (/sup 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.

  3. Molecular motors and their functions in plants

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.

    2001-01-01

    Molecular motors that hydrolyze ATP and use the derived energy to generate force are involved in a variety of diverse cellular functions. Genetic, biochemical, and cellular localization data have implicated motors in a variety of functions such as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. In non-plant systems three families of molecular motors (kinesins, dyneins, and myosins) have been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials intracellularly. During the last decade tremendous progress has been made in understanding the structure and function of various motors in animals. These studies are yielding interesting insights into the functions of molecular motors and the origin of different families of motors. Furthermore, the paradigm that motors bind cargo and move along cytoskeletal tracks does not explain the functions of some of the motors. Relatively little is known about the molecular motors and their roles in plants. In recent years, by using biochemical, cell biological, molecular, and genetic approaches a few molecular motors have been isolated and characterized from plants. These studies indicate that some of the motors in plants have novel features and regulatory mechanisms. The role of molecular motors in plant cell division, cell expansion, cytoplasmic streaming, cell-to-cell communication, membrane trafficking, and morphogenesis is beginning to be understood. Analyses of the Arabidopsis genome sequence database (51% of genome) with conserved motor domains of kinesin and myosin families indicates the presence of a large number (about 40) of molecular motors and the functions of many of these motors remain to be discovered. It is likely that many more motors with novel regulatory

  4. Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

    PubMed Central

    Weng, Mo

    2016-01-01

    Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT. PMID:26754645

  5. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  6. Myosin 1b functions as an effector of EphB signaling to control cell repulsion

    PubMed Central

    Prospéri, Marie-Thérèse; Lépine, Priscilla; Dingli, Florent; Paul-Gilloteaux, Perrine; Martin, René; Loew, Damarys; Knölker, Hans-Joachim

    2015-01-01

    Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion. PMID:26195670

  7. Motor Controllers

    NASA Technical Reports Server (NTRS)

    1984-01-01

    Kollmorgen Corporation's Mermaid II two person submersible is propeller-driven by a system of five DC brushless motors with new electronic controllers that originated in work performed in a NASA/DOE project managed by Lewis Research Center. A key feature of the system is electric commutation rather than mechanical commutation for converting AC current to DC.

  8. EPR and fluorescence depolarization studies on bovine cardiac myosin.

    PubMed

    Stone, D B; Mendelson, R A; Botts, J; Cheung, P H

    1981-09-01

    To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.

  9. Myosin Heavy Chain Composition of the Human Genioglossus Muscle

    PubMed Central

    Daugherty, Megan; Luo, Qingwei; Sokoloff, Alan J.

    2013-01-01

    Background The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation but whether this is accompanied by complex expression of muscle contractile proteins is not known. Purpose We tested for conventional myosin heavy chain MHCI, MHCIIA, MHCIIX, developmental MHCembryonic and MHCneonatal and unconventional MHCαcardiac, MHCextraocular and MHCslow tonic in antero-superior (GG-A) and posterior (GG-P) adult human GG. Method SDS-PAGE, Western blot and immunohistochemistry were used to describe MHC composition of GG-A and GG-P and the prevalence of muscle fiber MHC phenotypes in GG-A. Results: By SDS-PAGE, only conventional MHC are present with ranking from most to least prevalent MHCIIA>MHCI>MHCIIX in GG-A and MHCI>MHCIIA>MHCIIX in GG-P. By immunohistochemistry many muscle fibers contain MHCI, MHCIIA and MHCIIX but few contain developmental or unconventional MHC. GG-A is composed of five phenotypes (MHCIIA>MHCI-IIX>MHCI>MHCI-IIA>MHCIIX). Phenotypes MHCI, MHCIIA and MHCI-IIX account for 96% of muscle fibers. Conclusions Despite activation of GG during kinematically diverse behaviors and complex patterns of GG motor unit activity, the human GG is composed of conventional MHC isoforms and three primary MHC phenotypes. PMID:22337492

  10. Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

    PubMed Central

    Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M.; Ichimura, Taro

    2016-01-01

    Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery. PMID:27446684

  11. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    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.

  12. Comparison of neurological and functional outcomes after administration of granulocyte-colony-stimulating factor in motor-complete versus motor-incomplete postrehabilitated, chronic spinal cord injuries: a phase I/II study.

    PubMed

    Saberi, Hooshang; Derakhshanrad, Nazi; Yekaninejad, Mir Saeed

    2014-01-01

    Granulocyte-colony-stimulating factor (G-CSF) is a major growth factor in the activation and differentiation of granulocytes. This cytokine has been widely and safely employed in different disease conditions over many years. The administration of the growth factors in spinal cord injury (SCI) has been reported elsewhere; here we have tried to see the effect of SCI severity on the neurological outcomes after neuroprotective treatment for SCI with G-CSF. Seventy-four consecutive patients with SCI of at least 6 months' duration, with stable neurological status in the last 3 months, having informed consent for the treatment were included in the study. All the patients had undergone at least 3 months of standard rehabilitation. Patients were assessed by the American Spinal Injury Association (ASIA) scale, Spinal Cord Independence Measure (SCIM) III, and International Association of Neurorestoratology-Spinal Cord Injury Functional Rating Scale (IANR-SCIFRS) just before intervention and periodically until 6 months after subcutaneous administration of 5 µg/kg per day of G-CSF for 7 consecutive days. Multiple linear regression models were performed for statistical evaluation of lesion completeness and level of injury on changes in ASIA motor, light touch, pinprick, IANR-SCIFRS, and SCIM III scores, as a phase I/II comparative study. The study consisted of 52 motor-complete and 22 motor-incomplete SCI patients. There was no significant difference regarding age and sex, chronicity, and level of SCI between the two groups. Motor-incomplete patients had significantly more improvement in ASIA motor score compared to the motor-complete patients (7.68 scores, p < 0.001); also they had significant improvement in light touch (6.42 scores, p = 0.003) and pinprick sensory scores (4.89 scores, p = 0.011). Therefore, G-CSF administration in motor-incomplete SCIs is associated with significantly higher motor improvement, and also the higher the initial ASIA Impairment Scale

  13. Molecular Motors: A Theorist's Perspective

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.; Fisher, Michael E.

    2007-05-01

    Individual molecular motors, or motor proteins, are enzymatic molecules that convert chemical energy, typically obtained from the hydrolysis of ATP (adenosine triphosphate), into mechanical work and motion. Processive motor proteins, such as kinesin, dynein, and certain myosins, step unidirectionally along linear tracks, specifically microtubules and actin filaments, and play a crucial role in cellular transport processes, organization, and function. In this review some theoretical aspects of motor-protein dynamics are presented in the light of current experimental methods that enable the measurement of the biochemical and biomechanical properties on a single-molecule basis. After a brief discussion of continuum ratchet concepts, we focus on discrete kinetic and stochastic models that yield predictions for the mean velocity, V(F, [ATP], …), and other observables as a function of an imposed load force F, the ATP concentration, and other variables. The combination of appropriate theory with single-molecule observations should help uncover the mechanisms underlying motor-protein function.

  14. [C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin].

    PubMed

    Pronina, O E; Copeland, O; Marston, S; Borovikov, Iu S

    2006-01-01

    Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.

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

    PubMed

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

    2015-01-01

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

  16. A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip*

    PubMed Central

    Baboolal, Thomas G.; Mashanov, Gregory I.; Nenasheva, Tatiana A.; Peckham, Michelle; Molloy, Justin E.

    2016-01-01

    Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs. PMID:27566544

  17. Chara myosin and the energy of cytoplasmic streaming.

    PubMed

    Yamamoto, Keiichi; Shimada, Kiyo; Ito, Khoji; Hamada, Saeko; Ishijima, Akio; Tsuchiya, Takayoshi; Tazawa, Masashi

    2006-10-01

    Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.

  18. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Warshaw, David M.

    2016-01-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite’s intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein–tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  19. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments.

    PubMed

    Heaslip, Aoife T; Nelson, Shane R; Warshaw, David M

    2016-07-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  20. Compatibility of refrigerants and lubricants with motor materials under retrofit conditions. Final report, Volume II - data tables, high pressure refrigerants

    SciTech Connect

    Doerr, R.G.; Waite, T.D.

    1996-10-01

    Compatibility tests were conducted on motor materials to determine if exposure to the original refrigerant/mineral oil would affect compatibility of the motor materials after retrofit to the alternative refrigerant/lubricant. The motor materials were exposed at elevated temperature to the original refrigerant and mineral oil for 500 hours, followed by exposure to the alternative refrigerant and lubricant for 500 hours. Measurements were also taken after 168 and 336 hours. As a control, some samples were exposed to the original refrigerant/mineral oil for a total of 1000 hours.

  1. Nuclear myosin I regulates cell membrane tension.

    PubMed

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

    2016-08-02

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

  2. Nuclear myosin I regulates cell membrane tension

    PubMed Central

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

    2016-01-01

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

  3. Nuclear myosin I regulates cell membrane tension.

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2009-01-01

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

  5. The on-off switch in regulated myosins: different triggers but related mechanisms.

    PubMed

    Himmel, Daniel M; Mui, Suet; O'Neall-Hennessey, Elizabeth; Szent-Györgyi, Andrew G; Cohen, Carolyn

    2009-12-01

    In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long alpha-helical "heavy chain" stabilized by a "regulatory" light chain (RLC) and an "essential" light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca(2+) to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca(2+). Our results indicate that loss of Ca(2+) abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins. PMID:19769984

  6. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

    PubMed

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

  7. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

    PubMed Central

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P.

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections. PMID:22792440

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

    NASA Astrophysics Data System (ADS)

    Bryant, Zev

    2013-03-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2016-10-01

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

  11. Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

    PubMed Central

    Armel, Thomas Z.; Leinwand, Leslie A.

    2009-01-01

    Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, β-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the α-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule. PMID:19336582

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

    PubMed

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

    2016-10-01

    Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart. PMID:27568194

  13. Slow myosin in developing rat skeletal muscle

    PubMed Central

    1987-01-01

    Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC

  14. An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle.

    PubMed

    Smith, D A; Stephenson, D G

    2011-06-01

    The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl(-) binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small. PMID:21641314

  15. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    SciTech Connect

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony; Bernstein, Sanford I.

    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.

  16. Mechanosensing in Myosin Filament Solves a 60 Years Old Conflict in Skeletal Muscle Modeling between High Power Output and Slow Rise in Tension

    PubMed Central

    Marcucci, Lorenzo; Reggiani, Carlo

    2016-01-01

    Almost 60 years ago Andrew Huxley with his seminal paper (Huxley, 1957) laid the foundation of modern muscle modeling, linking chemical to mechanical events. He described mechanics and energetics of muscle contraction through the cyclical attachment and detachment of myosin motors to the actin filament with ad-hoc assumptions on the dependence of the rate constants on the strain of the myosin motors. That relatively simple hypothesis is still present in recent models, even though with several modifications to adapt the model to the different experimental constraints which became subsequently available. However, already in that paper, one controversial aspect of the model became clear. Relatively high attachment and detachment rates of myosin to the actin filament were needed to simulate the high power output at intermediate velocity of shortening. However, these rates were incompatible with the relatively slow rise in tension upon activation, despite the rise should be generated by the same rate functions. This discrepancy has not been fully solved till today, despite several hypotheses have been forwarded to reconcile the two aspects. Here, using a conventional muscle model, we show that the recently revealed mechanosensing mechanism of recruitment of myosin motors (Linari et al., 2015) can solve this long standing problem without any further ad-hoc hypotheses. PMID:27721796

  17. The optical trapping dumbbell assay for nonprocessive motors or motors that turn around filaments.

    PubMed

    Spudich, James A; Rice, Sarah E; Rock, Ronald S; Purcell, Thomas J; Warrick, Hans M

    2011-11-01

    In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.