Some aspects of the structural organization of the myofibril as revealed by antibody--staining methods.

FROM OBSERVATIONS OF FLUORESCENT ANTIBODY STAINING AND ANTIBODY STAINING IN ELECTRON MICROSCOPY, EVIDENCE IS PRESENTED FOR THE FOLLOWING: (a) Direct contact of the actin and myosin filaments occurs at all stages of contraction. This results in inhibition of antibody staining of the H-meromyosin portion of the myosin molecule in the region of overlap of the thin and thick filaments. (b) Small structural changes occur in the thick filaments during contraction. This leads to exposure of antigenic sites of the L-meromyosin portion of the myosin molecule. The accessibility of these antigenic sites is dependent upon the sarcomere length. (c) The M line is composed of a protein which is weakly bound to the center of the thick filament and is not actin, myosin, or tropomyosin. (d) Tropomyosin as well as actin is present in the I band. (e) If actin or tropomyosin is present in the Z line, it is masked and unavailable for staining with antibody.

The myosin filament. IV. Observation of the internal structural arrangement.

The subunit organization of the myosin filament of chicken striated muscle has been observed directly in cross-sections in electron microscopy. The organization consists of three centrally located and nine peripherally located subunits in a close-packed arrangement. This arrangement is that predicted by a previously derived model for the detailed molecular organization of the myosin filament (Pepe, 1966 a, 1967 a, 1971). Each subunit measures approximately 30 A in diameter and the center-to-center distance is approximately 37 A. If these measurements are considered to be on the high side, then they indicate that each subunit represents one myosin molecule. However, it is not possible to determine unequivocally whether one or two myosin molecules per subunit are present on the basis of this work.

The M band. Studies with fluorescent antibody staining.

The M band can be extracted from fibrils suspended in 5 mM Tris buffer, pH 8.0, for 15 min. The M band is completely removed only from fibrils of sarcomere lengths greater than 2.1 micro. Extraction does not alter the fluorescent antimyosin staining pattern of the A band, thus providing strong evidence that no alteration of the structural integrity of the thick filament has occurred. Fluorescent antibody staining of the M band of unextracted fibrils can be prevented specifically by absorbing the fluorescent antibody with extracted M band material prior to staining. This verifies the specificity of the extraction procedure.

Interaction between vertebrate skeletal and uterine muscle myosins and light meromyosins.

The specific contributions of this work may be summarized as follows: (a) No hybridization of uterine and skeletal myosin occurs at pH 6.0 although previous studies have shown that hybridization does occur at pH 6.5 (B. Kaminer et al. 1976. J. Mol. Biol. 100:379-386) or 7.0 (T. Pollard. 1975. J. Cell Biol. 67:93-104) (b) Hybridization of uterine and skeletal light meromyosins (LMM) occurs at pH 7.0 but not at pH 6.0, which is analogous to the hybridization of myosins. (c) In hybridized paracrystals there is a uniform distribution of both uterine and skeletal LMM molecules because all the paracrystals have only one axial repeat pattern. This makes it highly likely that in hybridized filaments the two myosins are also uniformly distributed throughout the filaments. (d) The 14-nm repeat of white bands observed in paracrystals of uterine LMM formed at pH 6.0, compared with the 14-nm repeat of dark bands observed with skeletal LMM under the same conditions, probably reflects differences in surface charge density along the different LMM molecules.

The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. I. Muscle structure at molting.

Rhodnius prolixus, a South American insect, molts five times in its development to an adult after emerging from the egg. Each molting cycle is triggered with a blood-meal. The ventral intersegmental abdominal muscles of Rhodnius develop during each molting cycle and are functional at molting. The fine structure of these fully developed muscles from fourth stage larval insects is studied. They have the characteristic structure of slow muscles. They have multiple motor nerve endings, and the myofibrils are poorly defined in cross-section. Longitudinal sections show long sarcomeres (8-10 micro), irregular Z-lines, and no apparent H zones. No M line is seen. Transverse sections through the A-band region show that each hexagonally arranged thick filament is surrounded by 12 thin filaments. Two thin filaments are shared by two neighboring thick filaments. The ratio of thin to thick filaments is 6:1. This structure is related to that found in vertebrate skeletal muscle and insect flight muscle.

The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. II. Muscle changes in preparation for molting.

The development of the ventral intersegmental abdominal muscles of Rhodnius prolixus is triggered by feeding. The early muscle (1 day after feeding) contains essentially nonstriated fibrils. However, in cross-sections, areas indicating early I bands, Z lines, and A bands can be recognized. Interdigitating thick and thin myofilaments do not assemble into a precise lattice until sometime between 4 and 5 days after feeding. As development continues, the number of fibrils increases, the region corresponding to the Z line increases in density, and the fibrils contain more recognizable striations. The newly formed fibrils broaden as myofilaments are added peripherally. At all stages throughout development, the ratio of thin to thick myofilaments is always 6:1. The formation of fibrils in the abdominal muscles of Rhodnius is different from that in chick embryo skeletal muscle. The major differences are that at all stages in Rhodnius there are (1) a constant ratio of thin to thick myofilaments, and (2) detectable Z-line material. Other findings in Rhodnius suggest (1) that fusion of mononucleated cells with the multinucleated muscle cell occurs, (2) that microtubules develop in the tendon cell concomitantly with development of myofibrils in the associated muscle cell, and (3) that filaments 55A in diameter aggregate into microtubules.

M band protein. Two components isolated from chicken breast muscle.

M band protein can be specifically extracted from fresh chicken breast muscle myofibrils suspended in 5 mM Tris-HCl pH 8.0. During discontinuous polyacrylamide gel electrophoresis the isolated protein separates into three bands which can be identified as two separate components (A, B) and a complex of the two. When partially purified fractions of the separated components are combined, an increase in the intensity of the band containing the complex can be shown. The polypeptide chain weights of the two components are 100,000 (A) and 40,000 (B) daltons as estimated by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Antibody prepared against total M band protein stains only the M band of the myofibril and is completely absorbed by M band protein. M band protein also absorbs the M band staining specifically from antibody which stains both I and M bands. Immunodiffusion data indicate that anti-M band is a mixture of two specific antibodies, one against each component.

Light meromyosin paracrystal formation.

STUDIES OF PARACRYSTAL FORMATION BY COLUMN PURIFIED LIGHT MEROMYOSIN (LMM) PREPARED IN A VARIETY OF WAYS LED TO THE FOLLOWING CONCLUSIONS: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.

The Z-band: 85,000-dalton amorphin and alpha-actinin and their relation to structure.

The conclusions arrived at as a result of this work can be summarized as follows: (a) We have found that there is an 85,000-dalton protein, which we have called 85K amorphin, associated with the Z-band of chicken pectoralis muscle myofibrils. We have isolated and purified this protein. It is not a structural component of the Z-filaments since it can be extracted completely without extraction of the Z-filaments. Extraction of 85K amorphin results in loss of specific staining of the Z-band with fluorescence specific anti-85K amorphin. (b) We have found that alpha-actinin is the structural component of the Z-filaments, since extraction of alpha-actinin is accompanied by loss of the Z-filament structure.

The myosin filament. XIII. The sensitivity of LMM assembly to Mg.ATP.

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.

LC2 involvement in the assembly of skeletal myosin filaments.

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.

Invertebrate myosin filament: subfilament arrangement in the wall of tubular filaments of insect flight muscles.

Transverse sections (100 to 140 nm thick) of the flight muscles of the fleshfly Phormia terrae-novae and the housefly Musca domestica were studied. The images of 56 tubular myosin filaments of the fleshfly and 62 filaments of the housefly were digitized and computer processed by rotational averaging. The rotational power spectra of more than 80% of the filaments showed peaks for 6-fold rotational frequency. The average of these images for each species showed a characteristic pattern consisting of 12 subunits arranged in six pairs around the wall of the filament. This pattern was enhanced by rotationally filtering the average images using the 6-fold components of the rotational power spectrum. On tilting individual images, the subunits behaved like rods perpendicular to the plane of the transverse section and they were therefore considered to be subfilaments essentially parallel to the long axis of the filament. The center-to-center spacing between the subfilaments of a pair is 2.8 nm, and the center-to-center spacing between the adjacent subfilaments of neighboring pairs is 4.0 nm. The observation of 12 subfilaments is consistent with a four-stranded helical arrangement of myosin cross-bridges on the surface of the filaments.

Antibody binding by native and denatured myosin and paramyosin.

By the techniques of immunodiffusion and fluorescent immunohistochemistry we show that antibodies to both the native and the SDS-denatured forms of the proteins, paramyosin and myosin, react with the native, SDS-denatured and glutaraldehyde-fixed forms of their respective antigens. Anti-denatured myosin also binds to both native and denatured forms of the proteolytic subfragments of myosin: globular subfragment-1 and alpha-helical LMM. Anti-native myosin, on the other hand, while able to bind to both native and denatured LMM or rod and to native and glutaraldehyde-fixed S-1, does not bind to SDS-denatured S-1.

Non-uniform staining of myofibril a bands by a monoclonal antibody to skeletal muscle myosin S1 heavy chain.

A monoclonal antibody specific for the S1 fragment of skeletal muscle myosin has been identified. The antibody does not inhibit actin-activated Mg2+-ATPase or K+-EDTA-activated ATPase of myosin, indicating that it is not related to the portion of the S1 which carries the ATPase activity. In the absence of relaxing medium, antibody binding to the myosin filament is restricted to narrow regions on each side of the bare zone region of the filament, and to a narrow region at the tapered ends of the filament. This restricted antibody binding is not altered by the attachment of the myosin cross-bridges to the actin filaments. In the presence of relaxing medium, antibody binding occurs along the entire length of the cross-bridge-bearing region of the filament. The restricted binding to only small regions of the filament in the absence of relaxing medium suggests that the molecular packing of the myosin in different portions of the filament may be different, resulting in differences in the availability of the antigenic site on the S1 for antibody binding. The change in availability of the antigenic sites along the filament in the presence of relaxing medium may reflect a perturbation in the molecular packing of the filament, or a conformational change resulting from the binding of MgATP, both of which could affect the availability of the antigenic sites on the S1 for antibody.

Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster, Homarus americanus.

The myosin filaments of the fast abdominal muscle of the lobster are about 2.7 microns long with a diameter of about 20 nm. They have a low density core in transverse sections except for a short portion in the middle of the filaments about 140 nm in length which is solid. In the solid region the diameter of the filaments is 25 nm. The wall of the filaments is made up of 12 subfilaments arranged in six pairs in a single layer around the wall. The spacing between the subfilaments of a pair is 3.4 nm and the spacing between successive pairs is 8.4 nm. An extra density is present on the inner surface of the wall of the filament along the entire length of the tubular portion of the filament. This density is always adherent to the wall and in serial transverse sections of the same filament its position changes from section to section without any apparent pattern to the change. No structural organization could be detected in this extra density.

Orientation of the backbone structure of myosin filaments in relaxed and rigor muscles of the housefly: evidence for non-equivalent crossbridge positions at the surface of thick filaments.

The orientation of the backbone structure of myosin filaments of relaxed and rigor fibers of the flight muscles of the housefly, Musca domestica, relative to the actin filaments has been investigated. In relaxed muscles 23% of the myosin filaments have gaps in the wall of their shaft located opposite the surrounding actin filaments, while in 77% the subfilament pairs of the wall are thus located. These are the expected values if the backbone orientation is random. In rigor muscles 40% of the thick filaments have their gaps opposite the actins and 60%, the subfilament pairs are opposite the actins. This increase in the percentage of filaments with gaps opposite the actins therefore results from binding of the crossbridges in rigor with change in rotational orientation of the backbone. The findings are related to a model of Beinbrech et al. (1988) in which two populations of crossbridges have been postulated: one originating at the surface of the thick filaments, the other coming from within the gap between the subfilament pairs.

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica.

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.

Substructures in the core of thick filaments: arrangement and number in relation to the paramyosin content of insect flight muscles.

Transverse sections (100-140 nm thick) of solid myosin filaments of the flight muscles of the honeybee, Apis mellifica, the fleshfly, Phormia terrae-novae and the waterbug, Lethocerus uhleri, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. The power spectra of the images of each of these filaments showed six-fold symmetry for the outer wall region and three-fold symmetry for the inner wall region. Images of the honeybee additionally showed three-fold symmetry for the center of the filament. Considering both paramyosin content of the myosin filaments and the results of the rotational filtering, we suggest the existence of 3 paramyosin strands in the myosin filaments of the fleshfly, 6 paramyosin strands in the honeybee filaments and 5 strands in the myosin filaments of the waterbug. In the case of the honeybee, the 3 paramyosin strands of the inner wall are positioned directly opposite the myosin subfilaments, while the 3 strands of the center seem to be arranged opposite the gaps between the myosin subfilaments. The paramyosin filaments of the fleshfly wobble between 2 myosin subfilaments, without loosing their three-fold symmetry arrangement in the inner wall. The 3 paramyosin strands in the inner wall of the waterbug myosin filaments are either arranged opposite the myosin subfilaments or opposite the gaps between the subfilaments. Finally, we were able to generate a 3-dimensional reconstruction of the myosin filament of the honeybee, showing the parallel arrangement of both, myosin subfilaments and paramyosin strands, relative to the long filament axis.

The myosin filament. X. Observation of nine subfilaments in transverse sections.

The molecular packing of the subfilaments in muscle thick filaments has been investigated by electron microscopy. Thin (80-100 nm) transverse sections of vertebrate skeletal muscle were cut, and 129 electron microscope images of thick filaments from 15 different areas including seven to ten images in each area were analyzed by computer image processing. The transverse sections were limited to the portion of the filaments between the bare zone and the C-protein bearing region. Of the 129 images, six were discarded because they were structurally disrupted, 17 did not show evidence for the presence of subfilaments from the autocorrelation function, and four did not show evidence for three-fold rotational symmetry from the power spectrum. The remaining 102 filaments all showed evidence for three-fold rotational symmetry, consistent with other available evidence (Pepe, 1982). From the analysis of these images by rotational filtering, we have found that the vertebrate skeletal myosin filament is made up of nine subfilaments and that the image appears to have trigonal symmetry. Of these subfilaments, six are arranged with a center-to-center spacing of about 4 nm and the other three on the surface of the filament are distorted from this arrangement. Three additional densities, which together with the other nine, correspond to the pattern of 12 densities previously observed in more highly selected images (Stewart et al., 1981; Pepe and Drucker, 1972) were observed in 5% of the images. Another pattern of nine subfilaments peripherally arranged around the circumference of the filament was observed occasionally. This latter image may represent the organization of the subfilaments in the bare zone region of the filament, resulting from sampling of individual filaments displaced longitudinally relative to the other filaments in the A-band.