Frog skeletal muscle thick filaments are three-stranded

A procedure has been developed for isolating and negatively staining vertebrate skeletal muscle thick filaments that preserves the arrangement of the myosin crossbridges. Electron micrographs of these filaments showed a clear periodicity associated with crossbridges with an axial repeat of 42.9 nm. Optical diffraction patterns of these images showed clear layer lines and were qualitatively similar to published x-ray diffraction patterns, except that the 1/14.3-nm meridional reflection was somewhat weaker. Computer image analysis of negatively stained images of these filaments has enabled the number of strands to be established unequivocally. Both reconstructed images from layer line data and analysis of the phases of the inner maxima of the first layer line are consistent only with a three-stranded structure and cannot be reconciled with either two- or four-stranded models.     

Rigor crossbridges are double-headed in fast muscle from crayfish

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.     

Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.

Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.     

The mammalian cardiac muscle thick filament: crossbridge arrangement

Although skeletal muscle thick filaments have been extensively studied, information on the structure of cardiac thick filaments is limited. Since cardiac muscle differs in many physiological properties from skeletal muscle it is important to elucidate the structure of the cardiac thick filament. The structure of isolated and negatively stained rabbit cardiac thick filaments has been analyzed from computed Fourier transforms and image analysis. The transforms are detailed, showing a strong set of layer lines corresponding to a 42.9 nm quasi-helical repeat. The presence of relatively strong "forbidden" meridional reflections not expected from ideal helical symmetry on the second, fourth, fifth, seventh, eighth, and tenth layer lines suggest that the crossbridge array is perturbed from ideal helical symmetry. Analysis of the phase differences for the primary reflections on the first layer line of transforms from 15 filaments showed an average difference of 170 degrees, close to the value of 180 degrees expected for an odd-stranded structure. Computer-filtered images of the isolated thick filaments unequivocally demonstrate a three-stranded arrangement of the crossbridges on the filaments and provide evidence that the crossbridge arrangement is axially perturbed from ideal helical symmetry.     

Structure and paramyosin content of tarantula thick filaments

Muscle fibers of the tarantula femur exhibit structural and biochemical characteristics similar to those of other long-sarcomere invertebrate muscles, having long A-bands and long thick filaments. 9-12 thin filaments surround each thick filament. Tarantula muscle has a paramyosin:myosin heavy chain molecular ratio of 0.31 +/- 0.079 SD. We studied the myosin cross-bridge arrangement on the surface of tarantula thick filaments on isolated, negatively stained, and unidirectionally metal-shadowed specimens by electron microscopy and optical diffraction and filtering and found it to be similar to that previously described for the thick filaments of muscle of the closely related chelicerate arthropod, Limulus. Cross-bridges are disposed in a four-stranded right-handed helical arrangement, with 14.5-nm axial spacing between successive levels of four bridges, and a helical repeat period every 43.5 nm. The orientation of cross-bridges on the surface of tarantula filaments is also likely to be very similar to that on Limulus filaments as suggested by the similarity between filtered images of the two types of filaments and the radial distance of the centers of mass of the cross-bridges from the surfaces of both types of filaments. Tarantula filaments, however, have smaller diameters than Limulus filaments, contain less paramyosin, and display structure that probably reflects the organization of the filament backbone which is not as apparent in images of Limulus filaments. We suggest that the similarities between Limulus and tarantula thick filaments may be governed, in part, by the close evolutionary relationship of the two species.     

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.     

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Electron microscopy and image analysis of myosin filaments from scallop striated muscle

Thick filaments have been isolated from the striated adductor muscle of the scallop and examined by electron microscopy after negative staining. Many filaments appear intact, and reveal a centrally located bare-zone and a well-defined helical surface array of myosin crossbridges characterized by a 145 A axial period and prominent helical tracks of pitch 480 A. Heavy-metal shadowing shows that these helices are right-handed. A small perturbation of alternate crossbridge levels produces an axial period of 290 A, which is most prominent in a region on either side of the bare-zone. Image analysis reveals that the crossbridge array has 7-fold rotational symmetry, one of the possibilities suggested by earlier X-ray diffraction studies of native filaments in scallop muscle. A low-resolution three-dimensional reconstruction shows elongated surface projections ("crossbridges") that probably represent unresolved pairs of myosin heads. They run almost parallel to the filament surface, but are slewed slightly from the axis so that they lie along the right-handed helical tracks of pitch 480 A. The connection to the filament backbone probably occurs at the end of the crossbridges nearer the bare-zone; thus, their sense of tilt appears to be opposite to that of rigor attachment to actin. The 290 A period arises from a different distribution of crossbridge density at alternate levels; in addition, there are weak connections between the top of one crossbridge and the bottom of the next, 145 A away. The prominence of the 290 A period near the bare-zone suggests that anti-parallel molecular interactions are mainly responsible for this perturbation.     

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.     

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

Myosin filament structure in vertebrate smooth muscle

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.     

Direct visualization of the myosin crossbridge helices on relaxed rabbit psoas thick filaments

Thick filaments in relaxed, quick-frozen and freeze-etched psoas myofibrils display a prominent helical pattern of projections repeating at 43 +/- 1 nm. These helices are right-handed, and measurement of the pitch angle indicates that the thick filaments are three-stranded. Each half-turn of a helix is composed of three to five projections, 11 to 12 nm in diameter. These projections probably represent individual myosin crossbridges. This is the first direct visualization of the crossbridge helices in vertebrate striated muscle filaments whose three-dimensional structure is preserved without chemical fixation.     

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.     

Equilibrium muscle crossbridge behavior: The interaction of myosin crossbridges with actin

The interaction of myosin crossbridges with actin under equilibrium conditions is reviewed. Similarities and differences between the weakly- and strongly-binding interactions of myosin crossbridges with actin filaments are discussed. A precise, narrow definition of weakly- binding crossbridges is given. It is postulated that the fundamental interaction of crossbridges with actin is that the crossbridge heads are mobile after attachment in the first case but not in the second. It is argued that because the weakly-binding crossbridge heads are mobile after attachment, the heads appear to function independently of each other. The lack of head mobility in attached strongly-binding crossbridges makes the strongly-binding crossbridge heads appear to act cooperatively. This model of the strongly-binding crossbridge gives an explanation for two important and otherwise unexplained observations. It explains why the rate constant of force decay after a small stretch is a sigmoidal function of nucleotide analogue concentration, and why, in the presence of analogues or in rigor, the rate constant of force decay after a small stretch is often significantly slower than the rate constant for myosin subfragment-1 detachment from actin in solution. The model of the weakly-binding crossbridge accurately describes the behavior of the myosin·ATP crossbridge.     

Structure of short thick filaments from Limulus muscle

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.     

Three-dimensional structure of the insect (Lethocerus) flight muscle M-band

The oval myosin filament profiles in transverse sections through the M-band of Lethocerus flight muscle are arranged in one of three orientations 60 degrees apart and point along the 11 directions of the hexagonal filament lattice. Relative orientations are not systematically related to give a superlattice structure, but neither are the orientations arranged completely randomly. In fact there is a nearly random structure with a slight bias towards adjacent filaments being identically oriented. This form of M-band structure is explained in terms of interactions between quasi-equivalent M-bridges. Its implications with regard to myosin crossbridge arrangement depend on the rotational symmetry of the crossbridge helix. For 6-stranded helices, 60 degrees rotations have no noticeable effect. However, in the case of the more likely 4-stranded structure, our results show that the crossbridge origins in the insect flight muscle A-band would be highly disordered. This disorder must be accounted for in interpreting both the flared-X crossbridge interactions seen in transverse sections of rigor insect flight muscle and the beautiful X-ray diffraction patterns from the same preparation. It is likely that in rigor insect muscle, some flared-Xs have the two heads of single myosin molecules interacting with two different actin filaments, whereas other flared-Xs have both of the myosin heads in one molecule interacting with the same actin filament.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

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.     

Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles,Revealed by Synchrotron X-Ray Fiber Diffraction

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.