Muscle Myosin Filaments Research Articles

Two Forms of Thick Filament in the Flight Muscle of Drosophila melanogaster.

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.

The effect of muscle ultrastructure on the force, displacement and work capacity of skeletal muscle.

Skeletal muscle powers animal movement through interactions between the contractile proteins, actin and myosin. Structural variation contributes greatly to the variation in mechanical performance observed across muscles. In vertebrates, gross structural variation occurs in the form of changes in the muscle cross-sectional area : fibre length ratio. This results in a trade-off between force and displacement capacity, leaving work capacity unaltered. Consequently, the maximum work per unit volume-the work density-is considered constant. Invertebrate muscle also varies in muscle ultrastructure, i.e. actin and myosin filament lengths. Increasing actin and myosin filament lengths increases force capacity, but the effect on muscle fibre displacement, and thus work, capacity is unclear. We use a sliding-filament muscle model to predict the effect of actin and myosin filament lengths on these mechanical parameters for both idealized sarcomeres with fixed actin : myosin length ratios, and for real sarcomeres with known filament lengths. Increasing actin and myosin filament lengths increases stress without reducing strain capacity. A muscle with longer actin and myosin filaments can generate larger force over the same displacement and has a higher work density, so seemingly bypassing an established trade-off. However, real sarcomeres deviate from the idealized length ratio suggesting unidentified constraints or selective pressures.

Near-atomic resolution structure of the Drosophila melanogaster flight muscle myosin filament

Near-atomic resolution structure of the Drosophila melanogaster flight muscle myosin filament

Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 Å Resolution Reveals New Details of Non-Myosin Proteins.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing.

The influence of biological sex in human skeletal muscle transcriptome during ageing.

Sex is a crucial biological variable, and influence of biological sex on the change of gene expression in ageing skeletal muscle has not yet been fully revealed. In this study, the mRNA expression profiles were obtained from the Gene Expression Omnibus database. Key genes were identified by differential expression analysis and weighted gene co-expression network analysis. The gene set enrichment analysis software and Molecular Signatures Database were used for functional and enrichment analysis. A protein-protein interaction network was constructed using STRING and visualized in Cytoscape. The results were compared between female and male subgroups. Differentially expressed genes and enriched pathways in different sex subgroups shared only limited similarities. The pathways enriched in the female subgroup were more similar to the pathways enriched in the older groups without taking sex difference into consideration. The pathways enriched in the female subgroup were more similar to the pathways enriched in the older groups without taking sex difference into consideration. The muscle myosin filament pathways were downregulated in the both aged female and male samples whereas transforming growth factor beta pathway and extracellular matrix-related pathways were upregulated. With muscle ageing, the metabolism-related pathways, protein synthesis and degradation pathways, results of predicted immune cell infiltration, and gene cluster associated with slow-type myofibers drastically different between the female and male subgroups. This finding may indicate that changes in muscle type with ageing may differ between the sexes in vastus lateralis muscle.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

STED analysis reveals the organization of nonmuscle muscle II, muscle myosin II, and F-actin in nascent myofibrils.

A three-step model has been proposed to describe myofibril assembly in vertebrate cardiac and skeletal muscle cells beginning with premyofibrils, followed by nascent myofibrils, and ending as mature myofibrils (reviewed in Sanger, Wang, et al. (2017). Assembly and maintenance of myofibrils in striated muscle. Handbook of Experimental Pharmacology 235, 39-75; Wang, Fan, (2020). Myofibril assembly and the roles of the ubiquitin proteasome system. Cytoskeleton 77, 456-479). Premyofibrils are composed of minisarcomeres that contain nonmuscle myosin II filaments interdigitating with actin filaments embedded at their barbed ends in muscle-specific alpha-actinin-rich Z-bodies. Sarcomeres in mature myofibrils have filaments of muscle myosin II that interact with actin filaments that are attached to muscle alpha-actinin in Z-bands. Nascent myofibrils, the transitional step between premyofibrils and mature myofibrils, possess two types of myosins II, that is, nonmuscle myosin II and muscle myosin II. The relationship of these two different myosins II in nascent myofibrils, however, is not clear. Stimulated emission depletion (STED) microscopic analyses of nascent myofibrils in both embryonic chick cardiomyocytes, and hiPSC-derived cardiomyocytes revealed that nonmuscle myosin II is in the middle of the nascent myofibril, surrounded by overlapping muscle myosin II filaments at the periphery, and non-striated filamentous actin is present in the nascent myofibril. These findings support the original three-step model of myofibril assembly proposed by Rhee, Sanger, and Sanger, (1994). The premyofibrils: Evidence for its role in myofibrillogenesis. Cell Motility and the Cytoskeleton 28, 1-24.

Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model

After decades of debate over the structure of smooth muscle myosin filaments, it is still unclear whether they are helical, as in all other muscle types, or square in shape. In both cases bipolar building units are proposed, but the deduced cross-bridge arrangements are fundamentally different. The opposite polarity of the adjusting longitudinal rows is proposed for the helical structure, while in the case of square filaments, or myosin ribbons, only their two faces are appositively polarized. Analysis of our unpublished archival data on light meromyosin (LMM) paracrystals and myosin rod assemblies as well as the filaments themselves indicated that the rods were assembled with a 6°–7° tilt angle from the rods’ longitudinal axis, in contrast to the lack of tilt in LMM, both exhibiting a 14.3 nm myosin periodicity. Optical diffraction analysis of EM images of the rod assemblies and those of intact myosin confirmed their helical architecture characterized by 28 nm residue translations, 172 nm repeats and 516 nm pitch. A detailed helical model of these filaments was elucidated with bipolar tetramer building units made of two polar trimers. The filaments elongate at their two ends in a head-to-head manner, enabling targeted cross-bridge polarity of the adjacent rows, in the form of a unique Boerdijk–Coxeter type helix, similar to that of collagen or desmin fibers, with the covalent links replaced by a head-to-head clasp.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

Defects in the Existing Theory of Skeletal Muscle Contraction and Postulation of a New Theory

In 1954 , two independent research teams, one consisting of Andrew F. Huxley and Rolf Niedergerke from the University of Cambridge, and the other consisting of Hugh Huxley and Jean Hanson from the Massachusetts Institute of Technology proposed the theory of skeletal muscle contraction [1]. They used electron microscopy to study the details of muscle filaments. The structure was studied in detail by then, but the mechanism of skeletal muscle contraction was not defined. Based on various assumptions about the actin and myosin filaments of muscle, later they postulated a theory called “sliding filament theory”. When this theory is scrutinized in detail, I find that there are a lot of defects in this theory, which I have pointed out and I have made an attempt to postulate a different mechanism for the skeletal muscle contraction.

Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

The load dependence and the force-velocity relation in intact myosin filaments from skeletal and smooth muscles.

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Abstract 107: Smooth Muscle α-actin Translocates to the Nucleus and Participates in Chromatin Remodeling at Smooth Muscle Contractile Gene Promoters

Smooth muscle cell specific α-actin (SMA, encoded by ACTA2 ), like other actins, polymerizes in the cytoplasm to form actin filaments that slide along smooth muscle myosin filaments to enact smooth muscle cell (SMC) contraction. Ubiquitously expressed β-actin polymerizes in filaments in the cytoplasm and also enters the nucleus and associates with various chromatin remodeling complexes. Nuclear β-actin has been shown to be important for the differentiation of a number of cell types, including neurons. We therefore asked whether SMA translocates to the nucleus and if nuclear SMA is critical for differentiation of smooth muscle cells. In explanted SMCs from human and mouse ascending aortas, cell fractionation and 2D gel electrophoresis identifies both SMA and β-actin in the nuclear isolates. Nuclear SMA but not β-actin accumulates with SMC differentiation driven by serum starvation and transforming growth factor β1 (TGFβ1) treatment. Chromatin immunoprecipitation with an SMA antibody reveals that the protein is bound to the promoters of SMC differentiation marker genes, including Acta2 , Cnn1, and Myh11 and that SMA is enriched over β-actin at these promoters with differentiation. Immunoprecipitation studies show that SMA binds specifically to the INO80 and the SWI/SNF chromatin remodeling complexes. In Acta2 -/- mouse SMCs, skeletal muscle α-actin (SKA) is ectopically expressed to compensate for the loss of SMA; SKA is in the nucleus and is enriched after TGFβ1 treatment, associating with both the INO80 and SWI/SNF complexes. Acta2 -/- SMCs are completely differentiated and respond normally to TGFβ1. However, in the mouse myoblast cell line C2C12, SKA is present in nuclear fractions but does not enrich with differentiation of these cells into myotubes, and is not associated with INO80 or SWI/SNF. We therefore propose that nuclear SMA is required for the remodeling of chromatin during the differentiation of SMCs, allowing contractile genes to become accessible for transcription, and that this remodeling is mediated by the involvement of SMA in the INO80 and SWI/SNF complexes These findings describe a novel role for SMA in the nucleus.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

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

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

A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, and Cardiac Muscle Myosin Filaments in Vitro

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/ton), where d equals step size and ton equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Force Produced by Smooth and Skeletal Muscle Myosin Filaments Measured with Micro-Fabricated Cantilevers

Force Produced by Smooth and Skeletal Muscle Myosin Filaments Measured with Micro-Fabricated Cantilevers