The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.     

Distribution of mass within native thick filaments of vertebrate skeletal muscle

The distribution of mass within the vertebrate skeletal thick filament has been determined by scanning transmission electron microscopy. Thick and thin filaments from fresh rabbit muscle were mixed with tobacco mosaic virus (TMV), fixed with formaldehyde, dried onto thin carbon films and viewed in a computer-linked microscope. Electron scattering data from both TMV and thick filaments were analysed with reference to the long axis of the particles so that the distribution of mass within the particles could be determined. While TMV appeared to be a uniform rod at the resolution employed (4.3 nm), the thick filament was clearly differentiated along its length. M-line remnants at the centre of the filament were flanked by regions of low mass per unit length, corresponding to the bare zone of the filament, and then by the more massive cross-bridge regions. The mass per unit length was approximately constant through most of the cross-bridge zone and declined at the filament tips, in a manner consistent with a constant number of myosin molecules per 14.3 nm interval (crown) throughout the cross-bridge zone. Fourier analysis of the data failed to detect the expected 43 nm periodicity of C-protein. The total mass of the thick filament was 184 Mdalton (s.e.m., 1.6 X 10(6); n = 70). The mass of adhering M-line proteins was highly variable but, on average, was about 4 Mdalton. The total mass of the filament and the mass distribution in the cross-bridge zone are consistent with three myosin molecules per crown.     

Electron microscope studies of thick filaments from vertebrate skeletal muscle.

Separated thick filaments have been prepared for electron microscopy by a method involving freeze-drying and shadowing. In the resulting filaments the individual heads of myosin molecules can be seen surrounding the filament shaft, which appears relatively smooth. Pairs of heads can frequently be seen to be emanating from a common origin. Myosin heads are found at distances up to 500 Å from the edge of the shaft.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

End-filaments: A new structural element of vertebrate skeletal muscle thick filaments

The use of low ionic strength buffers to dissociate separated thick filaments into three subfilaments is described. When the dissociation is performed in solution, rather than on an electron microscope grid, structures called end-filaments are observed where the subfilaments terminate. The end-filaments, only one of which is seen for every three subfilaments, are about 850 Å long, 50 Å wide and show transverse striations with a periodicity of 42 Å.     

Thin filaments of rabbit skeletal muscle are in helical register

Thin sections of rapidly frozen and freeze-substituted rabbit glycerinated muscle fibres loaded with myosin subfragment-1 were used to examine a three-dimensional arrangement of thin filaments in vertebrate skeletal muscle. Clearer images of the "arrowhead" structure were obtained when specimens were freeze-substituted first in a tannic acid solution and then in an OsO4 solution. The images obtained showed that the arrowheads were aligned laterally. This indicates that all the thin filaments have the same rotational orientation in a half sarcomere of rabbit skeletal muscle in the rigor state.     

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.     

Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle

Rabbit psoas muscle filaments, isolated in relaxing buffer from non-glycerinated muscle, have been applied to hydrophilic carbon films and stained with uranyl acetate. Electron micrographs were obtained under low-dose conditions to minimize specimen damage. Surrounding the filament backbone, except in the bare zone, is a fringe of clearly identifiable myosin heads. Frequently, both heads of individual myosin molecules are seen, and sometimes a section of the tail can be seen connecting the heads to the backbone. About half the expected number of heads can be counted, and they are uniformly distributed along the filament. The majority of heads appear curved. The remainder could be curved heads viewed from another aspect. Three times as many heads curve in a clockwise sense than in an anticlockwise sense, suggesting a preferential binding of one side of the head to the carbon film. The two heads of myosin molecules exhibit all the possible combinations of clockwise, anticlockwise and straight heads, and analysis of their relative frequencies suggests that the heads rotate freely and independently. The heads also adopt a wide range of angles of attachment to the tail. The lengths of heads cover a range of 14 to 26 nm, with a peak at 19 nm. The average maximum width is 6.5 nm. Both measurements are in excellent agreement with values for shadowed molecules. Since our data are from heads adsorbed to the film in relaxing conditions and the shadowed molecules were free of nucleotide, gross shape changes are not likely to be produced by nucleotide binding. The length of the link between the heads and the backbone was found to vary between 10 nm and 52 nm, with a broad peak at about 25 nm. Thus, the hinge point detected in the tail of isolated molecules was not usually the point from which the crossbridges swung out from the filament surface. The angle made by the link to the filament axis was between 20 degrees and 80 degrees, with a broad maximum around 45 degrees. These lengths and angles concur with our observation of an average limit of the crossbridges from the filament surface of 30 nm. This is sufficient to enable heads in the myofibril lattice to reach out beyond the nearest thin filament and should allow considerable flexibility for stereospecific binding to actin in active muscle.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

Thin filaments are not of uniform length in rat skeletal muscle

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3- , 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9- d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.     

Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments.

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Sequential disassembly of vertebrate muscle thick filaments

Native thick filaments from rabbit psoas muscle have been sequentially dissolved by incremental rises in salt concentration. Three quite separate stages of depolymerization can be detected; these presumably reflect constraints imposed on the disassembly process by variations in the packing of myosin and by the presence of other thick filament proteins.     

Structure of Limulus telson muscle thick filaments

Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab (Limulus polyphemus) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.     

H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle

With a view to obtaining a more complete view of the composition and structure of the thick filaments of vertebrate skeletal muscle, we have isolated and characterized two new myofibrillar components, H-protein and X-protein. These were purified by hydroxyapatite column chromatography of an impure C-protein preparation itself made from impure myosin extracted from rabbit back and leg muscles. H-protein is the protein responsible for band H on sodium dodecyl sulphate/polyacrylamide gel electrophoresis of crude myosin. X-protein, although present in such preparations in significant quantities, was not detected previously since it is difficult to resolve from C-protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Physical-chemical parameters have been determined for the new proteins and compared with those of C-protein. The apparent chain weight of H-protein estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis is 69,000, whereas that of X-protein (152,000) is only slightly greater than that of C-protein (140,000). The molecular weights of H- and X-proteins determined by sedimentation equilibrium centrifugation show that the molecules contain only a single polypeptide chain. The circular dichroism spectra indicate that the proteins have low alpha-helical contents. Both proteins, particularly H-protein, have a high proline content. Although X-protein is of similar chain weight to C-protein, the two show distinct differences in other properties. The sedimentation coefficient of X-protein is markedly lower than that of C-protein, suggesting X-protein is a more asymmetrical molecule. The amino acid compositions, although broadly similar, also show clear differences. Antibodies to H-protein, X-protein and C-protein have been raised in goats and shown not to cross-react.     

Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model

Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created.     

Bridgelike interconnections between thick filaments in stretched skeletal muscle fibers observed by the freeze-fracture method

The ultrastructure of frog semitendinosus muscle was explored using the freeze-fracture, deep-etch, rotary-shadowing technique. Mechanically skinned fibers were stretched to decrease or eliminate the overlap of thick and thin filaments before rapid freezing with liquid propane. In relaxed, contracting, and rigor fibers, a significant number of bridgelike interconnections, distinct from those observed in the M-region, were observed between adjacent thick filaments in the non-overlap region. Their half-length and diameter corresponded approximately to the known dimensions of the cross-bridge (or myosin S-1). The interconnection may thus be formed by the binding of two apposed cross-bridges projecting from adjacent thick filaments. Fixation with 0.5% glutaraldehyde for 5-10 min before freezing effectively preserved these structures. The results indicate that the interconnections are genuine structures that appear commonly in stretched muscle fibers. They may play a role in stabilizing the thick filament lattice, and possibly in the contractile process.