Myosin Motor Activity Research Articles

Dynamics of Non-Muscle Myosin II Organization into Stress Fibers and Contractile Networks

The cellular 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 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 them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin and stress 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.

Specific Charged Residues on the Myosin Heavy Chain (K368 and R406) Contribute to Head Interaction of Relaxed Smooth Muscle Myosin

Vertebrate smooth muscle contraction is regulated by phosphorylation of the myosin regulatory light chains. EM studies of smooth muscle myosin molecules suggest that in the dephosphorylated state, activity is switched off by asymmetric interactions between the two myosin heads, in which the “blocked” head is thought to be unable to bind to actin, while the “free” head has its ATPase activity inhibited. Comparative sequence analysis and molecular modeling suggest that the head interaction may be stabilized by charge attraction between D748 in the converter domain of the free head and K368 of the blocked head, and between R406 or K416 of the blocked head and E169 of the free head (Jung et al., MBC, 2008). To test these possibilities, we investigated the structure of expressed smooth muscle myosin and heavy meromyosin in which the charges at some of these sites was neutralized or reversed. While most of the mutants showed no obvious change in structure compared with wild type, HMM with charge reversal at both 368 and 406 showed fewer molecules with interacting heads when observed by metal shadowing. This was supported by a decrease in sedimentation coefficient (determined by analytical ultracentrifugation) and an increase in actin-activated ATPase activity of the mutant. Whole myosin with the same mutations formed filaments more readily than wild type, also consistent with these results. These observations suggest that charge interactions involving K368 and R406 play a role in stabilizing the head-head interaction of the inactive state. Interestingly, mutation of R403 in cardiac myosin (R406 in smooth muscle) increases motor activity of myosin and causes familial hypertrophic cardiomyopathy. Our results suggest that this could be in part a result of changes in head interaction.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca(2+) cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca(2+)/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca(2+) cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca(2+)(int), activating the Ca(2+)/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Molecules in motion: Michael Sheetz, James Spudich, and Ronald Vale receive the 2012 Albert Lasker Basic Medical Research Award

The Albert and Mary Lasker Foundation honors three pioneers in the field of molecular motor proteins, Michael Sheetz, James Spudich, and Ronald Vale (Figure ​(Figure1),1), for their contribution to uncovering how these proteins catalyze movement. Sheetz and Spudich developed the first biochemical assay to reconstitute myosin motor activity and showed that myosin and ATP alone were sufficient to direct transport along actin filaments. Fueled by this discovery, Vale and Sheetz examined transport in giant squid axon extracts and discovered a new motor, kinesin, that moves along microtubules. Cumulatively, their work opened the door for understanding how motor proteins drive transport of a host of molecules involved in numerous cellular processes, including cell polarity, cell division, cellular movement, and signal transduction. Figure 1 Ronald Vale (left), Michael Sheetz (center), and James Spudich (right) are the winners of the 2012 Albert Lasker Basic Medical Research Award.

Control of Myosin Motor Activity by the Reversible Alteration of Protein Structure for Application in a Bionanodevice

1Center for Diagnostic Nanosystems, Marshall University, Huntington, WV 2Department of Pharmacology, Physiology and Toxicology, Marshall University, Joan C. Edwards School of Medicine, Huntington, WV 3Department of Biological Sciences, Marshall University, Huntington, WV 4Department of Chemistry, Marshall University, Huntington, WV 5Department of Molecular and Cellular Pharmacology, Gunma University, Maebashi, Japan

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

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.

Influence of divalent cations on the cytoskeletal dynamics of K562 cells determined by nano-scale bead tracking

Cytoskeletal reorganization processes can be analyzed by studying the nanometer-scale spontaneous motion of beads bound to the cytoskeleton. The bead motion is determined by force fluctuations within the cytoskeletal network that originates from myosin motor activity and dynamic restructuring of cytoskeletal filaments. We investigated to what extend the spontaneous bead motion is influenced by the dynamics of the link between the bead and the cytoskeleton in the presence of divalent cations. Our data show that, when K562 cells expressing constitutively (alpha 5 beta 1) integrin and when stably transfected with (alpha v beta 3) integrin, spontaneous bead motion is dramatically affected by the presence of 1mM Mn2+ (integrin, activate state) compared to 1mM Ca2+/Mg2+ ions (integrin, inactive state). The directionality of the bead motion, which is influenced by the overall stability of the cytoskeletal network and by actomyosin-generated forces, is markedly different, whilst the persistence remained similar due to the specific binding of either Mn2+ or Mg2+/Ca2+ ions.

EMD57033 Acts as a Pharmacological Chaperone Stabilizing and Activating Myosin Motor Activity

Myosins play a crucial role for a wide range of physiological functions. The dysfunction or absence of myosin motors has been linked to diseases such as muscle wasting, heart failure, blood disorders, severe forms of senso-neuronal degeneration and cancer. Therefore, the development of selective and potent small-molecule myosin effectors holds the promise to open routes for the treatment of a wide range of disabling diseases. In this study, we show that the thiadiazinone derivative EMD57033 acts as a pharmacological chaperone for several members of the myosin family. Our results show that EMD57033 stimulates the actin-activated ATPase activity and motor activity of myosins from classes 2,5 and 6 up to 3-fold. The most potent interaction was observed with cardiac β-myosin. EMD57033 lowers the apparent second binding constant for F-actin binding in the presence of ATP from 31 μM (+/- 6 μM) to 4 μM (+/- 1 μM). The KM value for ATP remains unaffected by the drug. Furthermore, addition of 10 μM EMD57033 rescues the motor function of “aged” cardiac β-myosin preparations that display strongly reduced activity in the in vitro motility assay prior to addition of the drug. Structural studies indicate that EMD57033 binds in a pocket that is located between the N-terminal SH3-like subdomain and the motor domain-lever arm junction. The participation of the N-terminal SH3-like subdomain in EMD57033 binding is supported by the failure of class-1 myosins, which lack this domain, to productively interact with EMD57033.

Friction-Controlled Traction Force in Cell Adhesion

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.

Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity

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.

Phospholipid-dependent regulation of the motor activity of myosin X

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.

Unusual N-glycan Structures Required for Trafficking Toxoplasma gondii GAP50 to the Inner Membrane Complex Regulate Host Cell Entry Through Parasite Motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.

Regulation and function of the fission yeast myosins

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.

Ca 2+ Independent and Tail Dependent Regulation of the Motor Activity of Myosin X

Myosin X is involved in the actin cytoskeletal reorganization and protrusion of filopodia. Here we studied the molecular mechanism of regulation of myosin X. The actin-activated ATPase activity of M10Full was Ca2+ independent and significantly lower than that of M10HMM. The tail domain significantly inhibited the actin-activated ATPase activity of M10HMM regardless of Ca2+. The inhibition showed significant dependence on salt concentration, suggesting that the inhibition is dependent on ionic interaction between the tail domain and the head/neck domain of myosin X. The in vitro actin gliding velocity was markedly inhibited (4 fold) by the tail. These results suggest that the tail domain functions as an intra-molecular inhibitor of the myosin X motor function. The deletion of FERM domain abolished the inhibitory activity of the tail. On the other hand, deletion of the N-terminal PEST domain did not affect the inhibitory activity. Further truncation of the PH domain abolished the inhibitory activity of the tail. These results suggest that both the PH and FERM domains of the tail are required for the inhibition. On the other hand, the elimination of both IQ domains and the SAH/coiled-coil domain showed no effect on the tail induced inhibition. Furthermore, M10IQo co-immunoprecipitated with M10PH-FERM. The result indicated that the tail domain (PH-FERM) directly interacts with the motor domain to inhibit the motor activity. Electron microscopy revealed that the full-length myosin X molecules were monomeric, showing the wider molecules in low salt with ATP, while narrow molecules, similar head shapes to the M10HMM, in high salt. Our observation suggested that the tail domain folds backward to the head, such that it appeared to interact with the motor domain, and thus inhibits the motor activity of myosin X.(Supported by NIH).

Substrate-Ligand Friction Controls Traction Force in Cell Adhesion

Extracellular matrices determine cell fate decisions through the regulation of intracellular force and stress. The mechanical properties of these matrices like stiffness and ligand anchorage cause a distinct signaling behavior of adherent cells in respect to adhesion receptor forces. This process is known to be tightly linked to downstream kinase activation and epigenetic events including cell proliferation and differentiation. We report now on a new mechanism controlling traction forces in cell adhesion originating from sliding friction between adhesion ligands and the supporting material.Using polymer surfaces with a graded physicochemistry we were able to tune the non-covalent anchorage of adhesion ligands, namely fibronectin, to the materials surfaces. Traction force cytometry and in situ analysis of cell-driven ligand reorganization revealed a correlated dependence on the ligand-substrate anchorage. Ligand reorganization during the formation of fibrillar adhesions was characterized by a reaction-diffusion process with a surface mobility around 1e4 m/Ns. Based on these quantitative data we could describe the force-velocity equilibrium at the adhesion site with its extracellular and intracellular components, namely the nanoscale friction of adhesion ligands on the materials surface and the myosin motor activity at the actin stress fibers. The correlation of a linearized Tomlinson model of ligand friction on the materials surface with the characteristics of myosin motors in the actin stress fibers revealed a control mechanism of traction forces of around 1pN per receptor-ligand pair by the ligand-substrate friction.These findings elucidate a novel mechanism in force regulation at adhesion receptors, which is proposed to be highly relevant for cell behavior on natural and artificial scaffolds in vivo and in vitro, as many adhesion ligands are found non-covalently anchored to scaffold surfaces.

Active Patterning and Contractile Dynamics in Actin Networks Driven by Myosin Motors

Self-organized contractile arrays of actin filaments and myosin motors drive cell division, migration, and tissue morphogenesis. Biophysical studies have provided many insights into the mechanisms of force production by individual motor molecule. However, a mechanistic explanation of collective self-assembly into force-generating arrays is still lacking. We studied how the collective activity of myosin motors organizes actin filaments into contractile structures in a simplified model system devoid of biochemical regulation. We showed that myosin organizes actin into contractile arrays by a 3-stage process. First, the actin filaments mediate the formation of dense foci by active motor transport and motor coalescence. The myosin foci then accumulate actin filaments in a disordered cloud around them, and these actomyosin condensates finally merge into superaggregates by contractile coalescence. We propose that the origin of this multistage aggregation is the highly nonlinear load response of actin filaments, which can support large tensions but buckle already under picoNewton-compressive loads. Since the motor generated forces well exceed this buckling threshold, buckling is induced by the elastic resistance of connected actin networks to filament sliding. We furthermore mapped the spatiotemporal characteristics of the motor-induced contractility by tracking embedded particles, and found that myosin induces long-range, but localized, contractile fluctuations. The contractile dynamics and actomyosin superaggregates closely mimic observations in vivo. However, the localization and turnover of actomyosin arrays and directed cortical flows likely require an interplay of collective self-organization and biochemical regulation.

Mouse Models of Human MYH9-Related Diseases

Point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain IIA (NMHC IIA), underlie autosomal dominant syndromes in humans (incidence 1 in 500,000). The abnormalities can manifest as macrothrombocytopenia, granulocyte inclusions, progressive proteinuric renal disease, cataracts, and sensorineural deafness. To gain insight into the pathological mechanism of MYH9-related diseases in humans, we generated mouse models of three disease-associated mutations, Arg702Cys in the amino-terminal domain of NMHC IIA which controls myosin motor activity, and Asp1424Gln and Glu1841Lys in the carboxyl-terminal rod domain, which regulates filament formation.

The Epithelial Barrier Is Maintained by In Vivo Tight Junction Expansion During Pathologic Intestinal Epithelial Shedding

Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein-occludin or monomeric red fluorescent protein 1-ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.

Stochastic actin dynamics in lamellipodia reveal parameter space for cell type classification

The lamellipodium, a thin veil-like structure at the leading edge of motile cells, is fundamental for cell migration and growth. Orchestrated activities of membrane components and an underlying biopolymer film result in a controlled movement of the whole system. Dynamics in two-dimensional cell motility are primarily driven by the actively moving protein film in the lamellipodium. Polymerization of actin filaments at the leading edge, back-transport of the actin network due to myosin motor activity, depolymerization in the back, and diffusive transport of actin monomers to the front control these dynamics. The same molecular prerequisites for lamellipodial motion are found in most eukaryotic cells and can function independently of the cell body. Here we show that lamellipodial dynamics differ strongly in different cell types according to their function. Path finding neuronal growth cones display strong stochastic fluctuations, wound healing fibroblasts that locally migrate in tissues exhibit reduced fluctuations while fish keratocytes move highly persistently. Nevertheless, experimental analysis and computer simulations show that changes in the parameters for actin polymerization and retrograde actin transport alone are sufficient for the cell to utilize the same, highly adaptive machinery to display this rich variety of behaviors.

The recruitment of acetylated and unacetylated tropomyosin to distinct actin polymers permits the discrete regulation of specific myosins in fission yeast

Tropomyosin (Tm) is a conserved dimeric coiled-coil protein, which forms polymers that curl around actin filaments in order to regulate actomyosin function. Acetylation of the Tm N-terminal methionine strengthens end-to-end bonds, which enhances actin binding as well as the ability of Tm to regulate myosin motor activity in both muscle and non-muscle cells. In this study we explore the function of each Tm form within fission yeast cells. Electron microscopy and live cell imaging revealed that acetylated and unacetylated Tm associate with distinct actin structures within the cell, and that each form has a profound effect upon the shape and integrity of the polymeric actin filament. We show that, whereas Tm acetylation is required to regulate the in vivo motility of class II myosins, acetylated Tm had no effect on the motility of class I and V myosins. These findings illustrate a novel Tm-acetylation-state-dependent mechanism for regulating specific actomyosin cytoskeletal interactions.