Myosin Filament Assembly Research Articles

Moonwalking molecular machines: Unraveling the choreography of myosin filament assembly.

We have made tremendous progress in identifying the machines that shape the architecture of actin filaments. However, we know less about the mechanisms mediating myosin assembly at the supramolecular level. In this issue, Quintanilla et al. (https://doi.org/10.1083/jcb.202305023) provide important new insights into this process.

Limiting pool and actin architecture controls myosin cluster sizes in adherent cells

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.

Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis.

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.

Myosin 18Aα targets the guanine nucleotide exchange factor β-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is β-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines.Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺.Importantly, β-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted.Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with β-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of β-Pix's GEF activity.

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues. S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types.

Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the β-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

The sarcomeric cytoskeleton: from molecules to motion.

Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise.

Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.

Effects and mechanism of bufei yishen formula in a rat chronic obstructive pulmonary disease model.

Bufei Yishen Formula (BYF) has been used for centuries in Asia to effectively treat patients with chronic obstructive pulmonary disease (COPD). This study established a COPD animal model in rats, wherein three groups (control, COPD, and BYF) were used to evaluate the mechanism(s) and curative effect of BYF. Pulmonary function and histomorphology demonstrated that BYF had an evident effect on COPD. Gene microarray was then exploited to analyze the effects of BYF on COPD. ClueGO analysis of differentially expressed genes indicated that BYF improved COPD by regulating expression of interleukins, myosin filament assembly components, and mitochondrial electron transport-related molecules. Moreover, ELISA revealed that expression of several interleukins (IL1β, IL6, IL8, and IL10) was reduced in peripheral blood and bronchoalveolar lavage fluid by BYF treatment. It was concluded that BYF has therapeutic effects on COPD in rats through its effects on interleukin expression and/or secretion. Furthermore, pharmacological or targeted expression of two differentially expressed genes, F2R and Sprik1, might be useful in novel COPD therapies. This study provides the basis for mechanisms of BYF on COPD and new therapeutic drug targets.

Myosin filaments in smooth muscle cells do not have a constant length

Myosin molecules from smooth muscle and non-muscle cells are known to self-assemble into side-polar filaments in vitro. However, the in situ mechanism of filament assembly is not clear and the question of whether there is a unique length for myosin filaments in smooth muscle is still under debate. In this study we measured the lengths of 16,587 myosin filaments in three types of smooth muscle cells using serial electron microscopy (EM). Sheep airway and pulmonary arterial smooth muscle as well as rabbit carotid arterial smooth muscle were fixed for EM and serial ultra-thin (50-60 nm) sections were obtained. Myosin filaments were traced in consecutive sections to determine their lengths. The results indicate that there is not a single length for the myosin filaments; instead there is a wide variation in lengths. The plots of observation frequency versus myosin filament length follow an exponential decay pattern. Analysis suggests that in situ assembly of myosin filaments in smooth muscle is governed by random processes of linear polymerization and de-polymerization, and that the dynamic equilibrium of these processes determines the observed length distribution.

Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy

Understanding myofibrillogenesis is essential for elucidating heart muscle formation, development, and remodelling in response to physiological stimulation. Here, we report the dynamic assembly process of contractile myosin filaments onto myofibrils in a live cardiomyocyte culture during myofibrillogenesis. Utilizing a custom-built, two-photon excitation fluorescence and second harmonic generation imaging system equipped with an on-stage incubator, we observed new sarcomere additions in rat neonatal cardiomyocytes during 10 h of on-stage incubation. The new sarcomere additions occurred at the side of existing myofibrils, where we observed mature myofibrils acting as templates, or at the interstice of several separated myofibrils. During sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric alignment during maturation.

Myosin filament assembly requires a cluster of four positive residues located in the rod domain

Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins. Structured summary of protein interactionsWT LMMbinds to WT LMM by surface plasmon resonance (View Interaction)WT LMMbinds to CT2 LMM by surface plasmon resonance (View Interaction)WT LMMbinds to Alanine mutant LMM by surface plasmon resonance (View Interaction)WT LMM and WT LMMbind by light scattering (View Interaction)Alanine mutant LMM and Alanine mutant LMMbind by light scattering (View Interaction)WT LMM and Alanine mutant LMMbind by light scattering (View Interaction)

Asymmetric Mode of Ca2+-S100A4 Interaction with Nonmuscle Myosin IIA Generates Nanomolar Affinity Required for Filament Remodeling

SummaryFilament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca2+-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca2+-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (KD ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.

Gelsolin and Non-muscle Myosin IIA Interact to Mediate Calcium-regulated Collagen Phagocytosis

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca(2+) and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca(2+)](i), which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca(2+)](i) were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca(2+)](i), also required gelsolin. Ionomycin-induced increases of [Ca(2+)](i) overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca(2+)-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.

Cell spreading as a hydrodynamic process.

Many cell types have the ability to move themselves by crawling on extra-cellular matrices. Although cell motility is governed by actin and myosin filament assembly, the pattern of the movement follows the physical properties of the network ensemble average. The first step of motility, cell spreading on matrix substrates, involves a transition from round cells in suspension to polarized cells on substrates. Here we show that the spreading dynamics on 2D surfaces can be described as a hydrodynamic process. In particular, we show that the transition from isotropic spreading at early time to anisotropic spreading is reminiscent of the fingering instability observed in many spreading fluids. During cell spreading, the main driving force is the polymerization of actin filaments that push the membrane forward. From the equilibrium between the membrane force and the cytoskeleton, we derive a first order expression of the polymerization stress that reproduces the observed behavior. Our model also allows an interpretation of the effects of pharmacological agents altering the polymerization of actin. In particular we describe the influence of Cytochalasin D on the nucleation of the fingering instability.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

Cell spreading as a hydrodynamic process

Many cell types have the ability to move themselves by crawling on extra-cellular matrices. Although cell motility is governed by actin and myosin filament assembly, the pattern of the movement follows the physical properties of the network ensemble average. The first step of motility, cell spreading on matrix substrates, involves a transition from round cells in suspension to polarized cells on substrates. Here we show that the spreading dynamics on 2D surfaces can be described as a hydrodynamic process. In particular, we show that the transition from isotropic spreading at early time to anisotropic spreading is reminiscent of the fingering instability observed in many spreading fluids. During cell spreading, the main driving force is the polymerization of actin filaments that push the membrane forward. From the equilibrium between the membrane force and the cytoskeleton, we derive a first order expression of the polymerization stress that reproduces the observed behavior. Our model also allows an interpretation of the effects of pharmacological agents altering the polymerization of actin. In particular we describe the influence of Cytochalasin D on the nucleation of the fingering instability.