Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Three-Dimensional Structure of the M-region (Bare Zone) of Vertebrate Striated Muscle Myosin Filaments by Single-Particle Analysis

The rods of anti-parallel myosin molecules overlap at the centre of bipolar myosin filaments to produce an M-region (bare zone) that is free of myosin heads. Beyond the M-region edges, myosin molecules aggregate in a parallel fashion to yield the bridge regions of the myosin filaments. Adjacent myosin filaments in striated muscle A-bands are cross-linked by the M-band. Vertebrate striated muscle myosin filaments have a 3-fold rotational symmetry around their long axes. In addition, at the centre of the M-region, there are three 2-fold axes perpendicular to the filament long axis, giving the whole filament dihedral 32-point group symmetry. Here we describe the three-dimensional structure obtained by a single-particle analysis of the M-region of myosin filaments from goldfish skeletal muscle under relaxing conditions and as viewed in negative stain. This is the first single-particle reconstruction of isolated M-regions. The resulting three-dimensional reconstruction reveals details to about 55 Å resolution of the density distribution in the five main nonmyosin densities in the M-band (M6′, M4′, M1, M4 and M6) and in the myosin head crowns (P1, P2 and P3) at the M-region edges. The outermost crowns in the reconstruction were identified specifically by their close similarity to the corresponding crown levels in our previously published bridge region reconstructions. The packing of myosin molecules into the M-region structure is discussed, and some unidentified densities are highlighted.     

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.     

Arrangement of myosin heads on Limulus thick filaments

The two myosin heads with a single surface subunit on thick filaments from chelicerate arthropod muscle may originate from the same, or from axially sequential molecules, as suggested by three-dimensional reconstructions. The resolution attained in the reconstructions, however, does not permit one to distinguish unequivocally between these two possible arrangements. We examined the effect of 0.6 M KCl on relaxed thick filaments separated from Limulus muscle and filaments in which nearest myosin heads were cross-linked by the bifunctional agent, 3,3'-dithio-bis[3'(2')-O-[6-propionylamino)hexanoyl]adenosine 5'-triphosphate (bis22ATP), in the presence of vanadate (Vi). In high salt, surface myosin dissolved from both native, relaxed filaments and those exposed to 1-2 mM dithiothreitol after cross-linking, but was retained on filaments with cross-linked heads. Since bis22ATP must form intermolecular bonds between myosin heads within each subunit to prevent myosin solubilization in high salt, we conclude that each of these heads originates from a different myosin molecule, as was previously predicted by the reconstructions.     

Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sliding. In addition, although 3-D visualization of myosin crossbridges has been possible in rigor, it has been difficult for thick filaments actively interacting with thin filaments. In the current study, using three-dimensional reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for weak and strongly bound crossbridges on thin filaments. In relaxing conditions, tropomyosin was observed on the outer domain of actin, and thin filament interactions with thick filaments were rare. In contracting conditions, tropomyosin had moved to the inner domain of actin, and extra density, reflecting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now observed over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contraction occur in interacting thick and thin filaments, (2) that myosin-induced movement of tropomyosin in activated filaments requires strongly bound crossbridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of the crossbridge cycle.     

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.     

Influence of dispersion in myosin filament orientation and anisotropic filament contractions in smooth muscle

A new constitutive model for the biomechanical behaviour of smooth muscle tissue is proposed. The active muscle contraction is accomplished by the relative sliding between actin and myosin filaments, comprising contractile units in the smooth muscle cells. The orientation of the myosin filaments, and thereby the contractile units, are taken to exhibit a statistical dispersion around a preferred direction. The number of activated cross-bridges between the actin and myosin filaments governs the contractile force generated by the muscle and also the contraction speed. A strain-energy function is used to describe the mechanical behaviour of the smooth muscle tissue. Besides the active contractile apparatus, the mechanical model also incorporates a passive elastic part. The constitutive model was compared to histological and isometric tensile test results for smooth muscle tissue from swine carotid artery. In order to be able to predict the active stress at different muscle lengths, a filament dispersion significantly larger than the one observed experimentally was required. Furthermore, a comparison of the predicted active stress for a case of uniaxially oriented myosin filaments and a case of filaments with a dispersion based on the experimental histological data shows that the difference in generated stress is noticeable but limited. Thus, the results suggest that myosin filament dispersion alone cannot explain the increase in active muscle stress with increasing muscle stretch.     

3D structure of relaxed fish muscle myosin filaments by single particle analysis

To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) single particle analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head 'crowns' in the 429 A myosin filament repeat. The analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60 degrees , 60 degrees and 0 degrees , rather than the regular 40 degrees characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated.     

Actomyosin II interaction modulates cell cortex stability

Myosin II controls the viscoelastic behavior of actin filaments, interacting with actin in an energy-dependent manner. Replacing adenosine triphosphate with adenosine diphosphate changes actomyosin sliding to cross-linking. Rheological measurements show a 3-4-fold increase of the elastic portion G' in actin filaments when myosin II is present at a molar ratio r(MA)=1:200. This observation is supported by the demonstration of inactive myosin heads along actin filaments using atomic force microscopy.     

The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.     

Structural characterization of the binding of Myosin*ADP*Pi to actin in permeabilized rabbit psoas muscle

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

3D Structure of fish muscle myosin filaments

Myosin filaments isolated from goldfish (Carassius auratus) muscle under relaxing conditions and viewed in negative stain by electron microscopy have been subjected to 3D helical reconstruction to provide details of the myosin head arrangement in relaxed muscle. Previous X-ray diffraction studies of fish muscle (plaice) myosin filaments have suggested that the heads project a long way from the filament surface rather than lying down flat and that heads in a single myosin molecule tend to interact with each other rather than with heads from adjacent molecules. Evidence has also been presented that the head tilt is away from the M-band. Here we seek to confirm these conclusions using a totally independent method. By using 3D helical reconstruction of isolated myosin filaments the known perturbation of the head array in vertebrate muscles was inevitably averaged out. The 3D reconstruction was therefore compared with the X-ray model after it too had been helically averaged. The resulting images showed the same characteristic features: heads projecting out from the filament backbone to high radius and the motor domains at higher radius and further away from the M-band than the light-chain-binding neck domains (lever arms) of the heads.     

The mammalian cardiac muscle thick filament: backbone contributions to meridional reflections

Information about the structure of the vertebrate striated muscle thick filament backbone is important for understanding the arrangement of both the rod portion of the myosin molecule and the accessory proteins associated with the backbone region of the filament. Although models of the backbone have been proposed, direct data on the structure of the backbone is limited. In this study, we provide evidence that electron micrographs of isolated negatively stained cardiac thick filaments contain significant information about the filament backbone. Computed Fourier transforms from isolated cardiac thick filaments show meridional (or near meridional) reflections on the 10th and 11th layer lines that are particularly strong. Comparison of Fourier filtrations of the filaments that exclude, or include, these reflections, provide evidence that these reflections originate at least in part from a series of striations on the backbone at a approximately 4 nm spacing. The striations are likely to result either from the packing of the myosin rods, or from proteins such as titin associated with the filament backbone.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

Myosin Heads Contribute to the Maintenance of Filament Order in Relaxed Rabbit Muscle

Raising the temperature of rabbit skeletal muscle from ∼0°C to ∼20°C has been shown to enhance the helical organization of the myosin heads and to change the intensities of the 10 and 11 equatorial reflections. We show here by time-resolved x-ray diffraction combined with temperature jump that the movement of the heads to enhance the organized myosin helix occurs at the same fast rate as the change in the intensities of the equatorial reflections. However, model calculations indicate that the change in the equatorials cannot be explained simply in terms of the movement of myosin heads. Analysis of electron micrographs of transverse sections of relaxed muscle fibers cryofixed at ∼5°C and ∼35°C shows that in addition to the reorganization of the heads the thin and thick filaments are less constrained to their positions in the hexagonal filament lattice in the warm muscle than in the cold. Incorporating the changes in filament order in model calculations reconciles these with the observed changes in equatorial reflections. We suggest the thin filaments in the cold muscle are boxed into their positions by the thermal movement of the disordered myosin heads. In the warmer muscle, the packed-down heads leave the thin filaments more room to diffuse laterally.     

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 μM blebbistatin, disordering was at least 10 times slower. In the M·ADP state, myosin heads are also disordered. When blebbistatin was added to M·ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.     

Structure and nucleotide-dependent changes of thick filaments in relaxed and rigor plaice fin muscle

The myosin crossbridge array, positions of non-crossbridge densities on the backbone, and the A-band "end filaments" have been compared in chemically skinned, unfixed, uncryoprotected relaxed, and rigor plaice fin muscles using the freeze-fracture, deep-etch, rotary-shadowing technique. The images provide a direct demonstration of the helical packing of the myosin heads in situ in relaxed muscle and show rearrangements of the myosin heads, and possibly of other myosin filament proteins, when the heads lose ATP on going into rigor. In the H-zone these changes are consistent with crossbridge changes previously shown by others using freeze-substitution. In addition, new evidence is presented of protein rearrangements in the M-region (bare zone), associated with the transition from the relaxed to the rigor state, including a 27-nm increase in the apparent width of the M-region. This is interpreted as being mostly due to loss or rearrangement of a nonmyosin (M9) protein component at the M-region edge. The structure and titin periodicity of the end-filaments are described, as are suggestions of titin structure on the myosin filament backbone.     

Immunochemical localization of contractile proteins in mammalian meiotic chromosomes

Summary Smooth muscle heavy myosin and actin have been detected in mouse and rat meiotic chromosomes, by indirect immunofluorescence performed on testis cryostat sections and isolated germ cells. Both contractile proteins are detectable in the nuclei of meiotic cells during the first prophase. The appearance and disappearance time of myosin and actin, however, is not synchronous. While actin is visible in small spots from resting to late diplotene spermatocytes, myosin appears as filaments in the primary spermatocytes from the zygotene to the early stage of diplotene. The number of myosin filaments in the pachytene spermatocytes corresponds to the number of bivalent chromosomes, whereas actin spots constantly outnumber the pairing chromosomes by two units. These immunochemical observations suggest that the two contractile proteins are associated with the synaptonemal complex (SC). Myosin seems to be associated with the central region of the SC, while actin is present in its basal knob which is in connection with the nuclear membrane. The difference in number between myosin filaments and actin spots appears to be related to the peculiar behaviour of the pairing sex chromosomes. The presence of contractile proteins in the nuclei of primary spermatocytes seems to suggest that they might play a role in the process of pairing of homologous chromosomes.