The development of myofibrils in cultured muscle cells: A whole-mount and thin-section electron microscopic study

The development of myofibrils in cultured myotome cells from Xenopus embryos was studied with whole-mount and thin-section electron microscopy. For whole mount, the cells were grown on Formvar-coated grids, fixed, dehydrated, critical-point dried, and examined with a conventional (100 kV) or a high-voltage (1000 kV) electron microscope. Nonstriated bundles of 6- to 8-nm microfilaments, similar to stress fibers in nonmuscle cells, appear prior to nascent myofibrils. These bundles run the whole length of the cell and are inserted into the cell cortex. The transition from striated region to nonstriated region on a single nascent myofibril can be seen in both whole-mount and thin-section images. New sarcomeres appear to be added at the distal end of existing ones. Our data also indicate that these new sarcomeres are formed on a preexisting bundle of thin filaments. This suggests that the bundles of microfilaments are precursors to myofibrils. Evidence for this hypothesis came from the following observations. (1) Nascent myofibrils are anchored to the cell cortex via thin filaments similar to microfilament bundles. (2) Thin filaments in newly formed sarcomeres are often continuous through the middle of the A band. Later they break to form the H zone. (3) Thin filaments appear to be continuous through the developing Z band. Later they interact with the filaments in the Z band to form the staggered appearance.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

THE ULTRASTRUCTURE OF STRIATED MUSCLE AT VARIOUS SARCOMERE LENGTHS

1. Rest and equilibrium length muscle sarcomeres are composed of thin filaments (actin) which traverse the sarcomeres from the Z membranes up to the H band; at this level the filaments are considerably thicker and less numerous. 2. Shortening of muscle is associated with a transformation of thin into thick filaments in the A band. 3. These observations are discussed in terms of interaction of actin and myosin to form a supercoiled structure as the basis of contraction.     

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.     

Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti--actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of -actinin suggests that -actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Fine Structure of Cardiac Sarcomeres in the Black Widow Spider Latrodectus mactans

Fine structural characteristics of the cardiac muscle and its sarcomere organization in the black widow spider, Latrodectus mactans were examined using transmission electron microscopy. The arrangement of cardiac muscle fibers was quite similar to that of skeletal muscle fibers, but they branched off at the ends and formed multiple connections with adjacent cells. Each cell contained multiple myofibrils and an extensive dyadic sarcotubular system consisting of sarcoplasmic reticulum and T‐tubules. Thin and thick myofilaments were highly organized in regular repetitive arrays and formed contractile sarcomeres. Each repeating band unit of the sarcomere had three apparent striations, but the H‐zone and M‐lines were not prominent. Myofilaments were arranged into distinct sarcomeres defined by adjacent Z‐lines with relatively short lengths of 2.0 μm to 3.3 μm. Cross sections of the A‐band showed hexagon‐like arrangement of thick filaments, but the orbit of thin filaments around each thick filament was different from that seen in other vertebrates. Although each thick filament was surrounded by 12 thin filaments, the filament ratio of thin and thick myofilaments varied from 3:1 to 5:1 because thin filaments were shared by adjacent thick filaments.     

ELECTRON MICROSCOPIC STUDIES ON THE INDIRECT FLIGHT MUSCLES OF DROSOPHILA MELANOGASTER : I. Structure of the Myofibrils

The myofibrils in Drosophila have thick and thin types of myofilaments arranged in the hexagonal pattern described for Calliphora by Huxley and Hanson (15). The thick filaments, along most of their length in the A band, seem to be binary in structure, consisting of a dense cortex and a lighter medulla. In the H zone, however, they show more uniform density; lateral projections (bridges) also appear to be absent in this region. The M band has a varying number of granules (probably of glycogen) distributed between the myofilaments. The myofilaments on reaching the Z region appear to change their hexagonal arrangement and become connected to one another by Z filaments. The regular arrangement of the filaments found in most regions of the fibrils is not seen in the terminal sarcomeres of some flight muscles; the two types of filaments appear to be intermingled in an irregular pattern in these parts of the fibrils. The attachment of myofibrils to the cuticle through the epidermal cells is described.     

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Localized deposition of lead phosphate precipitate in glycerinated rabbit psoas muscle during ATP-induced contraction

The location of ATP-ase sites within sarcomeres was studied by allowing glycerinated rabbit psoas muscle to shorten under load in a solution containing ATP and lead nitrate. The lead phosphate precipitate formed during the contraction is located mainly within the A-band of shortened sarcomeres. In fibers glycerinated at rest length, the heaviest precipitate deposits are located at the center of the A-band, roughly corresponding to the double overlap zone, with lesser precipitate concentrations at the A-l boundary contraction bands. In fibers glycerinated at stretched length, shortened sarcomeres with heavy contraction bands at the A-l boundary and patent H-bands have heavy precipitate deposits over the contraction bands and no localization to the center of the A-band. The precipitate is thus seen to be most concentrated at those regions where one would expect to find the best contact between thick and thin filaments. Physiological experiments were performed. Evidence is presented that lead ions can substitute for calcium in the contraction process, altering the time course of contraction.     

STRUCTURE OF LIMULUS STRIATED MUSCLE : The Contractile Apparatus at Various Sarcomere Lengths

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 µm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 µm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.     

ELECTRON MICROSCOPIC STUDIES ON THE INDIRECT FLIGHT MUSCLES OFDROSOPHILAMELANOGASTER : II. Differentiation of Myofibrils

The differentiation of the indirect flight muscles was studied in the various pupal stages of Drosophila. Fibrillar material originates in the young basophilic myoblasts in the form of short myofilamants distributed irregularly near the cell membranes. The filaments later become grouped into bundles (fibrils). Certain "Z bodies" appear to be important during this process. The "Z bodies" may possibly be centriolar derivatives and are the precursors of the Z bands. The first formed fibrils (having about 30 thick myofilaments) are already divided into sarcomeres by Z bands. These sarcomeres, however, seem to be shorter than those of the adult fibrils.The H band differentiates in fibrils having about 40 thick myofilaments; the fibrils constrict in the middle of each sarcomere during this process. The individual myofibrils increase from about 0.3 µ to 1.5 µ in diameter during development, apparently by addition of new filaments on the periphery of the fibrils. The ribosomes seem to be the only cytoplasmic inclusions which are closely associated with these growing myofibrils. Disintegration of the plasma membranes limiting individual myoblasts was commonly seen during development of flight muscles, supporting the view that the multinuclear condition of the fibers of these muscles is due to fusion of myoblasts.     

Development of Vertebrate Skeletal Muscle

An investigation of developing skeletal muscle necessitatesthe study of three categories; the derivation of muscle cellsor fibers, myofilament synthesis and interactions, assemblyof myofilaments into functional sarcomeres of striated myofibrils.With few exceptions, skeletal muscle cells are of mesodermalorigin, and consist of rounded mononucleated cells which elongateand fuse with one another to become myotubes. Within the sarcoplasm,myofibrillar proteins are synthesized and grouped into interactingthick and thin filaments. Crude, non-striated myofibrils resultfrom linear arrangements of thick and thin filaments which arehorizontally aligned by the invaginating sarcotubular system.After Z-lines form, providing attachment sites for thin filaments,a typical banding pattern follows. The newly formed Z-linespull apart, followed by the attached thin filaments, and repeating"relaxed" sarcomeres are the resulting striated myofibrillarpattern.     

ULTRASTRUCTURE OF THE RESTING AND CONTRACTED STRIATED MUSCLE FIBER AT DIFFERENT DEGREES OF STRETCH

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 µ still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.     

CARDIAC MUSCLE OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS : I. Ultrastructure

The fine structure of the cardiac muscle of the horseshoe crab, Limulus polyphemus, has been studied with respect to the organization of its contractile material, and the structure of its organelles and the cell junctions. Longitudinal sections show long sarcomeres (5.37 µ at L_max), wide A bands (2.7 µ), irregular Z lines, no M line, and no apparent H zone. Transverse sections through the S zone of the A band show that each thick filament is ca. 180 A in diameter, is circular in profile with a center of low density, and is surrounded by an orbit of 9–12 thin filaments, each 60 A in diameter. Thick filaments are confined to the A band: thin filaments originate at the Z band, extend through the I band, and pass into the A band between the thick filaments. The sarcolemmal surface area is increased significantly by intercellular clefts. Extending into the fiber from these clefts and from the sarcolemma, T tubules pass into the fiber at the A-I level. Each fibril is enveloped by a profuse membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR occur at the A-I boundary where they make diadic contact with longitudinal branches of the T system. These branches also extend toward the Z, enlarge at the Z line, and pass into the next sarcomere. Infrequently noted were intercalated discs possessing terminal insertion and desmosome modifications, but lacking close junctions (fasciae occludentes). These structural details are compared with those of mammalian cardiac and invertebrate muscles.     

THE MYOFILAMENT ARRANGEMENT IN THE FEMORAL MUSCLE OF THE COCKROACH, LEUCOPHAEA MADERAE FABRICIUS

The structure of the femoral muscle of the cockroach, Leucophaea maderae, was investigated by light and electron microscopy. The several hundred fibers of either the extensor or flexor muscle are 20 to 40 µ in diameter in transverse sections and are subdivided into closely packed myofibrils. In glutaraldehyde-fixed and epoxy resin-embedded material of stretched fibers, the A band is about 4.5 µ long, the thin filaments are about 2.3 µ in length, the H zone and I band vary with the amount of stretch, and the M band is absent. The transverse sections of the filaments reveal in the area of a single overlap of thick and thin filaments an array of 10 to 12 thin filaments encircling each thick filament; whereas, in the area of double overlap in which the thin filaments interdigitate from opposite ends of the A band, the thin filaments show a twofold increase in number. The thick filament is approximately 205 to 185 A in diameter along most of its length, but at about 0.2 µ from the end it tapers to a point. Furthermore, some well oriented, very thin transverse sections show these filaments to have electron-transparent cores. The diameter of the thin filament is about 70 A. Transverse sections exhibit the sarcolemma invaginating clearly at regular intervals into the lateral regions of the A band. Three distinct types of mitochondria are associated with the muscle: an oval, an elongate, and a type with three processes. It is evident, in this muscle, that the sliding filament hypothesis is valid, and that perhaps the function of the extra thin filaments is to increase the tensile strength of the fiber and to create additional reactive sites between the thick and thin filaments. These sites are probably required for the functioning of the long sarcomeres.     

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.     

Disruption of Caenorhabditis elegans muscle structure and function caused by mutation of troponin I

Caenorhabditis elegans strains mutant for the unc-27 gene show abnormal locomotion and muscle structure. Experiments revealed that unc-27 is one of four C. elegans troponin I genes and that three mutant alleles truncate the protein: recessive and presumed null allele e155 terminates after nine codons; semidominant su142sd eliminates the inhibitory and C-terminal regions; and semidominant su195sd abbreviates the extreme C-terminus. Assays of in vivo muscular performance at high and low loads indicated that su142sd is most deleterious, with e155 least and su195sd intermediate. Microscopy revealed in mutant muscle a prevalent disorder of dense body positioning and a less well defined sarcomeric structure, with small islands of thin filaments interspersed within the overlap region of A bands and even within the H zone. The mutants' rigid paralysis and sarcomeric disarray are consistent with unregulated contraction of the sarcomeres, in which small portions of each myofibril shorten irregularly and independently of one another, thereby distorting the disposition of filaments. The exacerbated deficits of su142sd worms are compatible with involvement in vivo of the N-terminal portion of troponin I in enhancing force production, and the severe impairment associated with su195sd highlights importance of the extreme C-terminus in the protein's inhibitory function.