Myofibrillar M-band proteins represent constituents of native thick filaments,frayed filaments and bare zone assemblages

Summary A-segments, native thick filaments, frayed filaments, bare zone assemblages, as well as completely dissassembled and reassembled thick filaments from chicken pectoralis major were investigated for the presence of M_band proteins by the colloidal gold labelling technique. Specific polyclonal antibodies against the three M-band proteins identified to date, MM-creatine kinase, M-protein (165 kDa) and a 185 kDa protein myomesin, were prepared. Incubation with anti-M-protein and anti-myomesin antibodies resulted in heavy labelling of all thick filament types mentioned above, with the exception of the completely disassembled and reassembled thick filaments. In that case no labelling was detected with either antibody. In contrast, MM-creatine kinase which is an integral component of the intact M-band structure was detectable on isolated native thick filaments with lower frequency and to a variable extent. Also, bare zone assemblages were only rarely labelled by anti-MM-creatine kinase antibodies.This study shows that the cuff-like additional material which had previously been observed in the middle of the bare zone of isolated thick filaments represent remnants of all three M-band proteins, whereas the extra material in intact bare zone assemblages mainly consists of myomesin and M-protein, but not of MM-creatine kinase.Myomesin and M-line protein may be important for the assembly and structural maintenance of thick filaments as well as for anchoring of additional M-band proteins, e.g. MM-creatine kinase which is bound less tightly to thick filaments and, in accordance with earlier results, seems to represent within the M-band some of the prominent bridge-forming structures.     

Assembly of connectin (titin) in relation to myosin and α-actinin in cultured cardiac myocytes

Summary By using polyclonal and monoclonal antibodies against connectin (titin) which stain the A-I junctional area and the A-band domain (polyclonal anti-connectin and monoclonal 4C9) and the I-band domain (monoclonal SM1), the developmental relationship of this elastic protein with sarcomeric proteins, especially myosin and-actinin, was examined in embryonic chick cardiac myocytesin vitro under fluorescence microscopy. During premyofibril stages, I-Z-I proteins were detected first (-actinin dots and diffuse actin [phalloidin and anti-troponin C] staining), and later in these areas connectin and myosin dots appeared with nearly identical distribution. Somewhat later, phalloidin-positive nonstriated fibrils were observed in a straight course. They were always reactive with antibodies against a-actinin and troponin C, but unreactive or only weakly reactive with anticonnectin and anti-myosin. Initially,-actinin dots were aligned along these fibrils but did not form striations. As they aggregated to form Z-bands, connectin and myosin started to exhibit typical striations (doublets and A-bands, respectively). No difference in the staining pattern was observed with two kinds of monoclonal antibodies against different domains of connectin filaments (4C9 and SM1) at early phases. As myosin staining began to show clear A-bands, connectin epitopes became arranged in polarized positions. We conclude that primitive I-Z-I complexes appear prior to the assembly of connectin and myosin filaments and then connectin filaments, developing intimately and coordinately with myosin, become associated with the-actinin lines. Thus it appears that the putative elastic protein connectin plays some role in integrating myosin filaments with the preexisting I-Z-I brushes. The occasional absence of connectin and A-bands between two Z-bands, beyond both of which clear sarcomeres have been formed, indicates that connectin is not a preformed scaffold of myofibrils on which sarcomeric proteins accumulate.     

Developmental transitions in the myosin patterns of two fast muscles

Summary Transitions in myosin patterns were examined in situ by immunofluorescence in two fast muscles of the developing chicken, the pectoralis and the posterior latissimus dorsi. Myosin isoforms were localized using stage-specific monoclonal antibodies against the heavy chain of pectoralis myosin. Two antibodies (12C5 and 10H10) recognize adult and late embryonic myosin. They reacted weakly with both the pectoralis and posterior latissimus dorsi at 10 days in ovo, but intensely at 18 days in ovo. Both muscles were completely unreactive with an adult-specific antibody (5C3), indicating that the staining with 12C5 and 10H10 at 18 days in ovo reflects embryonic myosin. Thus two different embryonic isoforms are expressed sequentially in each muscle. Both 12C5 and 10H10 reacted weakly again with these muscles after hatching. The reappearance of a strong positive response to both antibodies, at 28 days in the pectoralis and after 60 days in the posterior latissimus dorsi, correlated well with the first appearance of a response to the adult-specific antibody, 5C3, signalling the beginning of the adult pattern. Both muscles reacted strongly with an antibody (5B4) specific for neonatal myosin between 18 days in ovo and 60 days after hatching. In the pectoralis, embryonic was replaced by neonatal myosin in most fibres by 14 days after hatching; by 28 days, both adult and neonatal myosin were expressed in most fibres; and in the adult, neonatal myosin was replaced entirely by the adult isoform. In contrast, many fibres in the posterior latissimus dorsi still expressed both embryonic and neonatal myosins up to at least 60 days post-hatch, and the remaining fibres expressed the neonatal isoform; the neonatal isoform was present in some fibres even in the adult posterior latissimus dorsi. We have therefore demonstrated in situ four different heavy chain isoforms in two different fast muscles. Early embryonic, late embryonic, neonatal and eventually adult isoforms are expressed in each muscle and more than one isoform often coexists in the same fibre.     

Effects of N-ethylmaleimide on the structure of skinned frog skeletal muscles

Summary The effects of N-ethylmaleimide (NEM) and other sulfhydryl modifiers on the structure of skinned frog skeletal muscles were studied using the X-ray diffraction technique. In sartorius muscle with full overlap between the thick and thin filaments, 0.1-1.0 mM NEM changed the intensity ratio of the (1,0) and (1,1) equatorial reflections from 4.35 to 0.72, and the (1,0) spacing of the hexagonal filament lattice from 40.4 to 41.4 nm. The axial X-ray diffraction pattern showed weak myosin layer-lines after the NEM treatment but enhancement of the actin layer-lines was not observed. In overstretched semitendinosus muscle, NEM did not affect the equatorial spacing but the myosin layer-lines were weakened. These results indicate that modification of myosin by NEM destroys the helical arrangement of myosin heads around the shaft of the thick filament and that when thin filaments are available, myosin heads move towards, and possibly bind to them. This binding is different from that in rigor since the ladder-like appearance of the higher actin layer-lines, which is typical of patterns from rigor muscles, was not observed. On removal of ATP after the NEM treatment, the diffraction pattern showed features characteristic of that from normal rigor muscles but no tension was produced. The pattern showed well-defined samplings on layer-lines in the small-angle region, indicating the presence of an extensive lattice order and exact axial alignment of the filaments. The first actin layer-line did not show samplings from the superlattice of the thick filaments, which are observed on the myosin layer-lines in patterns from resting muscles. This indicates that in NEM-treated rigor muscles the pattern of binding of myosin heads to the thin filaments is not influenced by the azimuthal orientation of the thick filament.     

Immunocytochemical electron microscopic study and Western blot analysis of myosin, paramyosin and miniparamyosin in the striated muscle of the fruit fly Drosophila melanogaster and in obliquely striated and smooth muscles of the earthworm Eisenia foetida

Miniparamyosin is a paramyosin isoform (55--60 kDa) that has been isolated in insects (Drosophila) and immunolocalized in several species of arthropods, molluscs, annelids and nematodes. In this study, the presence and distribution of this protein, in comparison with that of paramyosin and myosin, has been examined in the striated muscle (tergal depressor of trochanter) of Drosophila melanogaster, and the obliquely striated muscle (body wall) and the smooth muscle (outer layer of the pseudoheart) of the earthworm Eisenia foetida by means of immunocytochemical electron microscopic study and Western blot analysis miniparamyosin, paramyosin and myosin antibodies from Drosophila. In the striated muscle of D. melanogaster, the three proteins were immunolocalized along the length of the thick filaments (A- bands). The distribution of immunogold particles along these filaments was uniform. The relative proportions miniparamyosin/paramyosin/myosin (calculated by counting the number of immunogold particles) were: 1/10/68. In the obliquely striated muscle of E. foetida, immunoreactions to the three proteins were also found in the thick filaments, and the relative proportions miniparamyosin/paramyosin/myosin were 1/2.4/6.9. However, whereas the distribution of both myosin and miniparamyosin along the thick filament length was uniform, paramyosin immunolabelling was more abundant in the extremes of thick filaments (the outer zones of A-bands in the obliquely striated muscle), where the thick filaments become thinner than in the centre (the central zone of A-bands), where these filaments are thicker. The relative proportions of paramyosin in the outer and of paramyosin in the central zones of A-bands were 4/1. This irregular distribution of paramyosin along the thick filament length might be actual but it may also be explained by the fusiform shape of thick filaments in the earthworm: assuming that paramyosin is covered by myosin, paramyosin antigens would be more exposed in the tips than in the centre of thick filaments. If miniparamyosin is, in turn, covered by paramyosin, the exposure of miniparamyosin antigens would be low even in the tips of thick filaments, and this might explain the scanty immunoreaction observed for this protein and the absence of a higher number of immunogold particles in the extremes of thick filaments. The distribution of the three proteins in the earthworm smooth muscle was as in the obliquely striated muscle, although the proportions miniparamyosin/paramyosin/myosin were 1/1.5/5.2; this is, immunoreactions to paramyosin and miniparamyosin were lower than in the obliquely striated muscle     

Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly. First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.     

Location of paramyosin in relation to the subfilaments within the thick filaments of scallop striated muscle

Summary Myosin co-assembles with paramyosin in the thick filaments of invertebrate muscles. The molar ratio of the two proteins varies greatly but where sufficient paramyosin is present it forms the filament core with myosin arranged on its surface. In the fastest acting striated muscles, paramyosin is present in small amounts, and neither its location nor the nature of its interactions with myosin has previously been established. Antibodies to paramyosin have now been used in an attempt to locate the protein in thick filaments that have been isolated from the striated adductor muscle of the scallop and then frayed apart into their constituent subfilaments. Using a gold-conjugated secondary antibody, the location of paramyosin in relation to the subfilaments has been determined by electron microscopy of negatively stained samples. The labelling indicates that paramyosin extends throughout the length of the scallop filaments and appears to be associated with each subfilament, raising the possibility that in these filaments paramyosin may not be confined to a central core domain.     

Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies

Summary Monoclonal antibodies binding to epitopes in the rod portion of brush border myosin were used to study the mechanism of filament assembly and its role in myosin interaction with actin. The antibodies and their Fab fragments had specific effects on the size of the filaments assembledin vitro. Two antibodies (BM3 and BM4), directed against the tip of the myosin tail, completely inhibited myosin filament assembly. The other antibodies (BM1, BM2 and BM5), binding to other sites along the myosin rod, only partially blocked filament growth, and short filaments could be assembled. Thiophosphorylated brush border myosin filaments appeared slightly more stable to the effects of the antibodies than those composed of dephosphorylated myosin. Only one (BM3) of the antibodies which completely inhibited the assembly of new filaments was capable of disassembling preformed myosin filaments. The other antibody, BM4, partially disassembled filaments, leaving 0.2-m long cores, suggesting that polymerization in this myosin occurs by a biphasic Mechanism, I.e. the formation of a stable nucleus of antiparallely packed molecules, followed by elongation. The antibodies BM1 and BM2 bound to myosin filaments generating a regular transverse pattern with a 14-nm periodicity, and had little effect on the stability of these preformed filaments. Inhibition of filament formation and solubilization of the myosin by the antibodies appeared to be associated with inhibition of myosin interaction with actin, as measured by the actin-activated MgATPase activity. In the presence of the antibodies which completely inhibit filament assembly, we observed a decrease to 20% (BM4-Fab) and to 50% (BM3) of the control actin-activated myosin MgATPase activity, and this activity was kinetically different from that of the soluble myosin S1 fragment, suggesting that the rod has a profound effect on the kinetics of actomyosin interaction.     

Visualization of myosin exchange between synthetic thick filaments

Summary Exchange of myosin molecules between synthetic thick filaments was examined by fluorescence energy transfer and visualized by electron microscopy using streptavidin-gold to detect exchanged biotinylated myosin molecules. N-hydroxysuccinimidobiotin (NHS-biotin) was covalently linked to purified adult chicken pectoralis myosin to obtain assembly-competent biotinylated myosin molecules. Two distinct classes of synthetic filaments, distinguishable by length, were prepared. Biotinylated filaments (575±100 nm) were assembled by a quick dilution (QD) method and unlabelled filaments (1025±250 nm) were obtained by a sequential dilution (SD). The two filament population maintained their distinct length distributions even when mixed. To measure exchange, biotinylated short (QD) filaments were combined with unlabelled long (SD) filaments at a 15 ratio, sampled at varying times and the entry of biotinylated myosin into the previously unlabelled long filaments visualized by the addition of streptavidin-gold. The number of gold particles per micron was examined for fully biotinylated short filaments (<700 nm), unlabelled long filaments (>900 nm), and exchanged filaments. Equivalent binding of streptavidin-gold to the two filament types was detected by 60 min suggesting randomization of biotinylated monomers by this time. The precise location of streptavidin-gold sites on the long filaments was also measured. Although labeling was detected along the full length of the filaments, at the earliest time points (5 min) filament ends contained twice the number of gold particles as the filament centers. Approximately equivalent labeling along the entire length of the filaments was observed by 60 min. These results provide additional support for our earlier report of extensive myosin exchange between synthetic thick filaments and show that extensive exchange takes place rapidly along the full length of synthetic thick filaments.     

M-band structure,M-bridge interactions and contraction speed in vertebrate cardiac muscles

Summary Cardiac muscle M-band structures in several mammals (guinea pig, rabbit, rat and cow) and also from three teleosts (plaice, carp and roach), have been studied using electron microscopy and image processing. Axial structure seen in negatively stained isolated myofibrils or negatively stained cryo-sections shows the presence of five strong M-bridge lines (M6, M4, M1, M4 and M6) except in the case of the teleost M-bands in which the central M-line (M1) is absent, giving a four-line M-band. The M4 (M4) lines are consistently strong in all muscles, supporting the suggestion that bridges at this position are important for the structural integrity of the A-band myosin filament lattice. Across the vertebrate kingdom, cardiac M-band ultrastructure appears to correlate roughly with heartbeat frequency, just as in skeletal muscles it correlates with contraction speed, reinforcing the suggestion that some M-band components may have a significant physiological role. Apart from rat heart, which is relatively fast and has a conventional five-line M-band with M1 and M4 approximately equal, the rabbit, guinea pig and beef heart M-bands form a new 1+4 class; M1 is relatively very much stronger than M4.Transverse sections of the teleost (roach) cardiac A-band show a simple lattice arrangement of myosin filaments, just as teleost skeletal muscles. Almost all other vertebrate striated muscles, including mammalian heart muscles, have a statistical superlattice structure. The high degree of filament lattice order in teleost cardiac muscles indicates their potential usefulness for ultrastructural studies.It is shown that, in four-line M-bands in which the central (M1) M-bridges are missing, interactions at M4 (M4) are sufficient to define the different myosin filament orientations in simple lattice and superlattice A-bands. However the presence of M1 bridges may improve the axial order of the A-band.     

Ribosomes in the skeletal muscle filament lattice

The compartmentalization of myosin isoforms within a muscle cell (Gauthier: J. Cell Biol. 110:693–701, 1990) suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized. We therefore examined myofibrils by immunoelectron microscopy to determine whether ribosomes are associated with thick filaments under conditions in which new myosin can be identified. We used the embryonic chick anterior latissimus dorsi (ALD), a slow muscle that is induced, by curare, to synthesize a fast myosin isoform that is not normally present. Myosin was localized in situ, using a gold-labeled monoclonal antibody that recognizes the new isoform. The gold marker, as expected, was localized preferentially to the A band. There was an overall increase of fivefold in the number of gold particles per μm² of A band in the curare-treated compared to the normal ALD, indicating that the labeled isoform was largely newly formed. There was a corresponding preferential distribution of ribosomes at the A band, especially in the H-band region, and the number of ribosomes per μm² of A band was nearly twice as high in the curare-treated as in the normal muscle. Ribosomes were located between thick filaments, often aligned in rows. We conclude that ribosomes are located within the filament lattice, and therefore that they are available for local myosin synthesis. © 1993 Wiley-Liss Inc.     

Stretchin-klp, a novel Drosophila indirect flight muscle protein, has both myosin dependent and independent isoforms

Stretchin-klp is a newly described protein in Drosophila indirect flight muscles (IFM) that migrates on SDS gels as two distinct components of approximately 225 and 231 kD. Although the larger isoform is IFM specific, the smaller stretchin-klp isoform is expressed not only in IFM, but also in wild-type tissues of the adult head, abdomen and thorax from which the IFM has been removed. It is not detected, however, in jump or leg muscles. Probes derived from a cDNA encoding part of stretchin-klp hybridize with a 6.7 kb mRNA. Stretchin-klp is one of several putative products of the Stretchin-Myosin light chain kinase gene and is predicted to have multiple immunoglobulin domains arranged in tandem pairs separated by variable length spacers. Polyclonal antibodies directed against the expressed peptide of the stretchin-klp cDNA label the IFM myofibril A-band, though not its central and lateral regions. Analyses of IFM mutants indicate that the larger stretchin-klp isoform is myosin dependent. Although the normal adult myosin filament or the ‘headless’ myosin rod is sufficient for accumulation of both the large and small stretchin-klp isoforms, loss of myosin, or substitution of the adult rod with an embryonic one in IFM prevents the larger isoform from being formed or stabilized. During development stretchin-klp is first detected at pupal stage p8, when myofibrils are being constructed. These studies suggest that this newly identified protein is a major component of the Drosophila IFM thick filament.     

Radial stability of the actomyosin filament lattice in isolated skeletal myofibrils studied using atomic force microscopy

The radial stability of the actomyosin filament lattice in skeletal myofibrils was examined by using atomic force microscopy. The diameter and the radial stiffness of the A-band region were examined based on force–distance curves obtained for single myofibrils adsorbed onto cover slips and compressed with the tip of a cantilever and with the Dextran treatment. The results obtained indicated that the A-band is composed of a couple of stiffness components having a rigid core-like component. It was further clarified that these radial components changed the thickness as well as the stiffness depending on the physiological condition of myofibrils. Notably, by decreasing the ionic strength, the diameter of the A-band region became greatly shrunken, but the rigid core-like component thickened, indicating that the electrostatic force distinctly affects the radial structure of actomyosin filament components. The results obtained were analyzed based on the elementary structures of the filament lattice composed of cross-bridges, thin filaments and thick filament backbones. It was clarified that the actomyosin filament lattice is radially deformable greatly and that (1), under mild compression, the filament lattice is stabilized primarily by the interactions of myosin heads with thin filaments and thick filament backbones, and (2), under severe compression, the electrostatic repulsive interactions between thin filaments and thick filament backbones became predominant.     

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases

Summary The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity,v (=-dL/dt where L is the length of thick filaments at timet), increased with increasing KCl concentration (0.225 to 0.5 m), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25° C), micromolar concentrations of Ca²⁺ or sarcomere length (2.4 and 2.75 m). Over a wide range of filament length, the dissociation velocity could be expressed byv ₀exp(L), wherev ₀ and are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.     

Characterization of connectin-like proteins of obliquely striated muscle of a polychaete (Annelida)

Summary In obliquely striated muscle of polychaete, Neanthes sp., three kinds of connectin (titin)-like high molecular weight proteins, 4000 kDa, 1200 kDa and 700 kDa, were detected by SDS gel electrophoresis and immunoblots using antibodies to vertebrate skeletal muscle connectin and antiserum to the protein in question. The 700 kDa protein was isolated and characterized as a sheet-rich filament 170 nm long and 4 nm wide. Using polyclonal antibodies to the 700 kDa protein, the binding of the immunogold to the thick filament was only demonstrated in high ionic strength relaxing solution which solubilized some myosin. This observation suggested that the 700 kDa protein was localized below the layers of myosin in the thick filament and this localization is different from that of twitchin of C. elegans bodywall muscle that is on the surface of thick filament. The 4000 kDa protein was identified as a very thin filament linking the thick filament to the dense body. The very thin filaments were visualized in gelsolin-treated actin filament-free fibres. The 1200 kDa protein was located in the periphery of the dense body. A model of the elastic filament in polychaete bodywall muscle is presented.     

Thick filament assembly occurs after the formation of a cytoskeletal scaffold

The development of myofibrils involves the formation of contractile filaments and their assembly into the strikingly regular structure of the sarcomere. We analysed this assembly process in cultured human skeletal muscle cells and in rat neonatal cardiomyocytes by immunofluorescence microscopy using antibodies directed against cytoskeletal and contractile proteins. In particular, the question in which temporal order the respective proteins are integrated into developing sarcomeres was addressed. Although sarcomeric myosin heavy chain is expressed as one of the first myofibrillar proteins, its characteristic A band arrangement is reached at a very late stage. In contrast, titin, then myomesin and finally C-protein (MyBP-C) gradually form a regularly arranged scaffold on stress fiber-like structures (SFLS), on non-striated myofibrils (NSMF) and on nascent striated myofibrils (naSMF). Immediately subsequent to the completion of sarcomere cytoskeleton formation, the labeling pattern of myosin changes from the continuous staining of SFLS to the periodic staining characteristic for mature myofibrils. This series of events can be seen most clearly in the skeletal muscle cell cultures and – probably due to a faster developmental progression – less well in cardiomyocytes. We therefore conclude that the correct assembly of a cytoskeletal scaffold is a prerequisite for correct thick filament assembly and for the integration of the contractile apparatus into the myofibril.     

Morphological changes and recovery process in the tenotomized soleus muscles of the rat.

Tenotomized soleus muscles of adult rats were analyzed morphologically and biochemically with special reference to the recovery process. Light microscopic observations of semi-thin sections showed that the characteristic central core lesion was most extensive at 1 week after tenotomy and began to diminish in extent at 2 weeks until no trace of lesion could be seen by 6th week, as confirmed by thin-section electron microscopy. Three phases of changes in the cross-sectional area of muscle fibers after tenotomy were demonstrated by morphometry: phase I designated as the initial increase up to the 3rd day, phase II as the progressive decrease until the 4th week, and phase III as the recovery to normal or even hypertrophy. In electron microscopy, the earliest alteration of myofibrils was recognized at 3 days after tenotomy. The Z discs showed a wavy or zigzag profile with frequent longitudinal splitting of myofibrils. From the 2nd week on, muscle fibers underwent a process of recovery, replacing the central core lesion with new myofibrils in which a reassembly of thick filaments into bundles of thin filaments took place, with Z discs being aligned adjacent to the peripheral complete myofibrils. In SDS-polyacrylamide gel electrophoresis, the molar ratio of myosin to actin diminished markedly as the central core lesion developed and gradually returned to normal with time, correlating well with the loss and subsequent reassembly of thick filaments.     

High-voltage electron microscopy of crossbridge interactions in striated muscle

Summary In order to investigate the geometry of the interactions which myosin molecules make with actin filaments we have studied thick (0.2–0.5 µm) transverse sections of striated muscles in the 1 MeV electron microscope at Imperial College. Sections obtained from fixed relaxed frog sartorius muscle and both fixed relaxed and fixed rigor insect flight muscles, show regular electron opaque features between the thick and thin filament profiles. These are thought to be the overlapping images of the many levels of myosin heads that occur in such sections. From the appearances of these images, together with studies of thin transverse sections, it appears that of the possible interactions which one myosin molecule can make, namely that its two component heads interact with the same thin filament or with two different thin filaments, it is the former interaction (both heads on the same filament) which is predominant. Nevertheless appearances have been seen similar to those expected if an interaction of one molecule with two thin filaments occurs. It is concluded that both single filament and two filament interactions can occur depending on the steric convenience of the available actin subunits, but that the single filament interaction occurs in the majority of cases in the muscle states we have studied.Finally it is shown that the myosin filament profiles seen in thick transverse sections may be a very misleading guide to thick filament structure because of the influence which the myosin crossbridges have on the appearance of the profiles.     

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 ± 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 ± 1.4 nm, and intermediate filaments with mean diameters of 9.3 ± 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/μm² at 6 days to 43 intermediate filaments/μm² at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.