High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding are only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.

A mechanogenetic role for the actomyosin complex in branching morphogenesis of epithelial organs.

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the crucial genes required for the distribution of epithelial progenitor cells were regulated by YAP and TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.

The Structure of Acto-Myosin.

After several decades studying different acto-myosin complexes at lower and intermediate resolution - limited by the electron microscope instrumentation available then - recent advances in imaging technology have been crucial for obtaining a number of excellent high-resolution 3D reconstructions from cryo electron microscopy. The resolution level reached now is about 3-4 Å, which allows unambiguous model building of filamentous actin on its own as well as that of actin filaments decorated with strongly bound myosin variants. The interface between actin and the myosin motor domain can now be described in detail, and the function of parts of the interface (such as, e.g., the cardiomyopathy loop) can be understood in a mechanistical way. Most recently, reconstructions of actin filaments decorated with different myosins, which show a strongly bound acto-myosin complex also in the presence of the nucleotide ADP, have become available. The comparison of these structures with the nucleotide-free Rigor state provide the first mechanistic description of force sensing. An open question is still the initial interaction of the motor domain of myosin with the actin filament. Such weakly interacting states have so far not been the subject of microscopical studies, even though high-resolution structures would be needed to shed light on the initial steps of phosphate release and power stroke initiation.

Afadin regulates actomyosin organization through αE-catenin at adherens junctions.

Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with ß-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with ß-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with ß-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with ß-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with ß-catenin and E-cadherin at adherens junctions.

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

Unconventional Imaging Methods to Capture Transient Structures during Actomyosin Interaction.

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

Nuclear displacement and fluorescence recovery after photobleaching (FRAP) assays to study division site placement and cytokinesis in fission yeast.

Cytokinesis is an essential cellular event that completes the cell division cycle. It begins with the assembly of an actomyosin contractile ring that undergoes constriction concomitant with the septum formation to divide the cell in two. Placement of the septum at the right position is important to ensure fidelity of the division process. In fission yeast, the medially placed nucleus is a major spatial cue to position the site of division. In this chapter, we describe a simple synthetic biology-based approach to displace the nucleus and study the consequence on division site positioning. We also describe how to perform fluorescence recovery after photobleaching to follow the dynamics of cytokinetic proteins at defined time points by live-cell microscopy.

Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution.

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated during a complex mechanochemical reaction cycle. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins, a high-resolution structure of the F-actin­myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release) states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges.

It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley's 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255-318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197-218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d=8 nm gave an ADP release rate 8.5-fold faster and ton (myosin's attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V=N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V=L/ton rather than d/ton, where L is related to the length of myosin's subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d=11.1 nm. Therefore, in contrast with Huxley's model, we conclude that V0 is limited by the actin-myosin attachment rate.

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity.

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front-back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front-back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines.

Septum development in Neurospora crassa: the septal actomyosin tangle.

Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, BUD-4, and CHS-1. In chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (SAT) assembly, 2) contractile actomyosin ring (CAR) formation, 3) CAR constriction together with plasma membrane ingrowth and cell wall construction. Septation began with the assembly of a conspicuous tangle of cortical actin cables (SAT) in the septation site >5 min before plasma membrane ingrowth. Tropomyosin and myosin were detected as components of the SAT from the outset. The SAT gradually condensed to form a proto-CAR that preceded CAR formation. During septum development, the contractile actomyosin ring remained associated with the advancing edge of the septum. Formin and BUD-4 were recruited during the transition from SAT to CAR and CHS-1 appeared two min before CAR constriction. Actin patches containing fimbrin were observed surrounding the ingrowing septum, an indication of endocytic activity. Although the trigger of SAT assembly remains unclear, the regularity of septation both in space and time gives us reason to believe that the initiation of the septation process is integrated with the mechanisms that control both the cell cycle and the overall growth of hyphae, despite the asynchronous nature of mitosis in N. crassa.

Actomyosin sliding is attenuated in contractile biomimetic cortices.

Myosin II motors embedded within the actin cortex generate contractile forces to modulate cell shape in essential behaviors, including polarization, migration, and division. In sarcomeres, myosin II-mediated sliding of antiparallel F-actin is tightly coupled to myofibril contraction. By contrast, cortical F-actin is highly disordered in polarity, orientation, and length. How the disordered nature of the actin cortex affects actin and myosin movements and resultant contraction is unknown. Here we reconstitute a model cortex in vitro to monitor the relative movements of actin and myosin under conditions that promote or abrogate network contraction. In weakly contractile networks, myosin can translocate large distances across stationary F-actin. By contrast, the extent of relative actomyosin sliding is attenuated during contraction. Thus actomyosin sliding efficiently drives contraction in actomyosin networks despite the high degree of disorder. These results are consistent with the nominal degree of relative actomyosin movement observed in actomyosin assemblies in nonmuscle cells.

Reconstitution of contractile actomyosin arrays.

Networks and bundles comprised of F-actin and myosin II generate contractile forces used to drive morphogenic processes in both muscle and nonmuscle cells. To elucidate the minimal requirements for contractility and the mechanisms underlying their contractility, model systems reconstituted from a known set of purified proteins in vitro are needed. Here, we describe two experimental protocols our lab has developed to reconstitute 1D bundles and quasi-2D networks of actomyosin that are amenable to quantitative biophysical measurement. These assays have enabled our discovery of the mechanisms of contractility in disordered actomyosin assemblies and of a mechanical feedback between contraction and F-actin severing.

Motor-driven marginal band coiling promotes cell shape change during platelet activation.

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Radial stability of the actomyosin filament lattice in isolated skeletal myofibrils studied using atomic force microscopy.

The radial stability of the actomyosin filament lattice in skeletal myofibrils was examined by using atomic force microscopy. The diameter and the radial stiffness of the A-band region were examined based on force-distance curves obtained for single myofibrils adsorbed onto cover slips and compressed with the tip of a cantilever and with the Dextran treatment. The results obtained indicated that the A-band is composed of a couple of stiffness components having a rigid core-like component. It was further clarified that these radial components changed the thickness as well as the stiffness depending on the physiological condition of myofibrils. Notably, by decreasing the ionic strength, the diameter of the A-band region became greatly shrunken, but the rigid core-like component thickened, indicating that the electrostatic force distinctly affects the radial structure of actomyosin filament components. The results obtained were analyzed based on the elementary structures of the filament lattice composed of cross-bridges, thin filaments and thick filament backbones. It was clarified that the actomyosin filament lattice is radially deformable greatly and that (1), under mild compression, the filament lattice is stabilized primarily by the interactions of myosin heads with thin filaments and thick filament backbones, and (2), under severe compression, the electrostatic repulsive interactions between thin filaments and thick filament backbones became predominant.

Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain.

In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis.

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

We used transient phosphorescence anisotropy to detect the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin strongly bound to single-headed fragments of muscle myosin subfragment 1 (S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at substoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than with muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects: muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly and muscle myosin binds more slowly to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound erythrosin iodoacetamide: while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex are determined by the isoform of the bound myosin in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

Site-directed spectroscopic probes of actomyosin structural dynamics.

Spectroscopy of myosin and actin has entered a golden age. High-resolution crystal structures of isolated actin and myosin have been used to construct detailed models for the dynamic actomyosin interactions that move muscle. Improved protein mutagenesis and expression technologies have facilitated site-directed labeling with fluorescent and spin probes. Spectroscopic instrumentation has achieved impressive advances in sensitivity and resolution. Here we highlight the contributions of site-directed spectroscopic probes to understanding the structural dynamics of myosin II and its actin complexes in solution and muscle fibers. We emphasize studies that probe directly the movements of structural elements within the myosin catalytic and light-chain domains, and changes in the dynamics of both actin and myosin due to their alternating strong and weak interactions in the ATPase cycle. A moving picture emerges in which single biochemical states produce multiple structural states, and transitions between states of order and dynamic disorder power the actomyosin engine.