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.     

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.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

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).     

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.     

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.     

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments

Long, thick filaments (greater than 4.0 micrometer) rapidly and gently isolated from fresh, unstimulated Limulus muscle by an improved procedure have been examined by electron microscopy and optical diffraction. Images of negatively stained filaments appear highly periodic with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrographs show a wealth of detail and are consistent with a myosin helical repeat of 43.8 nm, similar to that observed by x-ray diffraction. Analysis of the optical diffraction patterns, in conjunction with the appearance in electron micrographs of the filaments, supports a model for the filament in which the myosin cross-bridges are arranged on a four-stranded helix, with 12 cross-bridges per turn or each helix, thus giving an axial repeat every third level of cross-bridges (43.8 nm).     

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.     

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.     

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.     

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

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.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.     

Optical polarization properties of the diffraction spectra from single fibers of skeletal muscle.

The diffraction spectra of laser light from single fibers of skeletal muscle exhibit a large degree of optical depolarization. When the linearly polarized incident laser source is oriented at polarization angles between 0 less than theta less than pi/2 rad with respect to the fiber axis, the diffracted light is elliptically polarized. These results show that the phase angle of the ellipse rotates by as much as 20 degrees when the fiber is stretched from 2.4 to 3.8 microns. To further ascertain that the observed phenomenon is diffraction related, an experiment monitoring the spectra of scattered light in between diffraction orders showed this signal to be significantly more linearly polarized. These results suggest that the degree of elliptical polarization of the diffraction spectra is a sensitive probe of A-band dynamics, including changes of the anisotropic S-2 elements.     

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.     

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.