The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary Changes in the contractile apparatus of denervated rat soleus muscles were investigated during the course of reinnervation.As observed earlier, in the course of denervation atrophy the ratio of myosin to actin filaments decreases because myosin filaments disappear faster than actin filaments (Jakubiec-Puka et al. 1981 a). After reinnervation the amount of myosin filaments and myosin heavy chains (myosin HC) in the muscle increased during the first few days; the increment of actin content was negligible. The proportion of myosin HC to actin remained lower than normal for about 30 days. The excess of actin filaments frequently observed in the newly-formed myofibrils reflects this disproportion.The results show a lability of myosin and suggest some cytoskeletal role for actin filaments.     

Supercontraction in crayfish muscle: Correlation with a peculiar actin localization

Summary Crayfish muscle, like muscles from some other invertebrates, can supercontract. This muscle shortening is characterized by an overlap of thin filaments with crossing of thick filaments through the Z discs. In intact muscle cells, supercontraction does not seem to induce irreversible structural modifications in the tissue.Isolated crayfish myofibrils in the relaxed state cannot be distinguished from vertebrate myofibrils under light microscope, either by phase contrast or by immunofluorescence, with antiactin antibodies, actin being localized in the I bands. However, when isolated crayfish myofibrils are supercontracted, irreversible dammage occurs, most thin filaments being lost. Actin becomes then hardly detectable, being visible, by immunofluorescence, either in the Z discs or evenly distributed in the whole myofibril.During myofibril supercontraction, high amounts of denatured actin, become soluble as shown by SDS-PAGE, by double immunodiffusion, and by DNAse inhibition.Abbreviations used in the text EGTA ethyleneglycol-bis (-aminoethyl ether)-N, N-tetraacetic acid - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - TEMED N, N, N, N-tetramethylenediamine - TRIS Tris (hydroxymethyl) aminomethane A preliminary report on this work was presented at the meeting of the Union of Swiss Societies for Experimental Biology, Davos, 1978 (Benzonana et al., 1978)     

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin

Summary Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against - and -actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the sarcomeric proteins -actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of sarcomeric proteins in regard to myofibrillogenesis.     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: anisoptera)

Summary The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 . The A-band length is about 3 . The myofibrils consist of thick (250 Å) and thin (85 Å) filaments. Each thick filament is surrounded by 9–12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed.The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Development of myofibrils in the gizzard of chicken embryos

Summary The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, -actinin, and desmin, in smooth muscle cells of chicken gizzard at various stages of embryogenesis were investigated by immunofluorescence-labeling of enzyme-dispersed cells cultured up to three hours. These muscle proteins, except some part of myosin, were organized into fibrous structures as soon as synthesis and accumulation of proteins started. As for myosin, a considerable amount of it was dispersed in soluble cytoplasm as well. On the other hand, Ca⁺⁺-dependent contractility was detected with detergent-extracted myoblasts and glycerinated tissue from embryos older than 7 days. Although the nascent myofibrils bear a resemblance to stress fibers, the former could be distinguished from the latter by their high stability in dispersed, spherical cells. The above findings, therefore, show that the synthesis of contractile proteins is followed by immediate assembly of them into functional myofibrils without undergoing any intermediate structure. Based on these findings, the mechanism of myofibril formation in developing smooth muscle cells is discussed.     

The fine structure of differentiating muscle in the salamander tail

Summary Thin methacrylate sections of developing tails of Amblystoma opacum larvae were examined in the electron microscope and a series of stages in the differentiation of the myotome musculature was reconstructed from electron micrographs and earlier light microscopic studies of living muscle. The earliest muscle cell precursor that can be clearly identified is a round or oval cell with abundant cytoplasm containing scattered myofilaments and free ribonucleoprotein granules, but little endoplasmic reticulum. These cells sometimes form a syncytium and they may also be fused with adjacent formed muscle fibers by lateral processes. Nuclei are large and nucleoli are prominent. This cell, called a myoblast here, is distinctly different in its appearance from the adjacent mesenchymal cells which have abundant granular endoplasmic reticulum. The earliest myofilaments are of both the thick and thin varieties and are distributed in a disorganized fashion in the cytoplasm. These filaments are similar to the actin and myosin filaments described by Huxley and they are present in the cytoplasm at an earlier stage of differentiation than heretofore suspected from light microscopy studies. The first myofibrils are a heterogeneous combination of thick and thin filaments and dense Z bands and are not homogeneous as so many light microscopists have contended. As development progresses, cross striations become more orderly and definitive sarcomeres are formed. Thereafter, new myofilaments and Z bands seem to be added to the lateral surfaces and distal ends of existing myofibrils.Free ribonucleoprotein granules are a prominent part of the myoblast cytoplasm and are found in close association with the differentiating myofilaments in all stages of development. In early muscle fibers and some of the formed fibers, similar granules are often concentrated in the I bands. A theory of myofilament differentiation based on current concepts of the role of ribonucleoprotein in protein synthesis is presented in the discussion. Stages in myofibril formation and possible relationships of the filaments in developing muscle cells to other types of cytoplasmic filaments are also discussed.Supported by grant C-5196 from the United States Public Health Service.     

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.     

Ultrastructure of the heart muscle cells of the cuttlefish Rossia macrosoma (Delle Chiaje) (Mollusca: Cephalopoda)

Summary The muscle cells of the ventricle, the branchial heart and the branchial heart appendages of Rossia macrosoma (Delle Chiaje) are studied. The ventricle myocardium has three muscle layers, while the other two organs exhibit a loose arrangement of muscle cells. The muscle cells of the ventricle, the branchial heart and the branchial heart appendages are similar in structure. The nuclei are surrounded by myofibrils. In the myofibrils A-, I- and discontinuous Z-bands are seen. The diameters of the thick filaments are 300–400Å, their length varies from 1.7 to 3.9 . Thin filaments have a diameter of approximately 85Å. The ratio between thick and thin filaments is roughly 1 to 11.The SR runs mostly as a longitudinal network within the myofibrils. A few short T-tubules are observed in the Z-regions. Peripheral and internal couplings exist. The latter are few in number.Intercalated discs are small and rarely observed. They have been found in all three organs. A difference in the function of these organs is not reflected in the ultrastructure of the intercalated discs. These discs are often of the interdigitating type with interfibrillar junctions and unspecialized regions. Peripheral couplings are seen at the unspecialized regions. The intercalar surfaces of the muscle cells shoulder off into the lateral surface, and the transition between the two surfaces is not a sharp one. Attachment plaques are found scattered over the whole sarcolemma.     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Fine structure of muscles in the tickHyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

Skeletal and visceral muscles are distinguished in the unfed nymphHyalomma (Hyalomma) dromedarii according to position, structure and function. The skeletal muscles include the capitulum, dorsoventral and leg oblique muscles. Their muscle fibres have the striated pattern of successive sarcomeres whose thick myosin filaments are surrounded by orbitals of up to 12 thin actin filaments. The cell membrane invaginates into tubular system (T) extending deeply into the sarcoplasm and closely associated to cisternae of sarcoplasmic reticulum (SR). The T and SR forming two-membered dyads are considered to be the main route of calcium ions whose movements are synchronized with the motor impulse to control contraction and relaxation in most muscles. Two types of skeletal muscle fibres are recognized, and are suggested as representing different physiological phases.In the visceral-muscle fibres investing tick internal organs, the actin and myosin filaments are slightly interrupted, and the T and SR are well demonstrated. Both skeletal and visceral muscles are invaginated by tracheoles and innervated by nerve-axons containing synaptic vesicles.     

Quantitative analysis of muscle breakdown during starvation in the marine flatfish Pleuronectes platessa

Summary The present study describes the effects of starvation for a duration of four months on the ultrastructure of skeletal muscles from the marine flatfish (Pleuronectes platessa L.). Starvation is associated with a decrease in resting metabolic rate from 20.1±2.2 to 11.6±1.5mg-O₂/kg/h (P<0.05) and muscle wasting. Median fibre size fell from 700 m² to 500 m² in intermediate (fast oxidative) and from 1,800 m² to 600 m² in starved, white (fast-glycolytic) muscle fibres. In contrast, median fibre size in red (slow oxidative) muscle remained within the range 300–400 m². The fraction of red fibre volume occupied by myofibrils (58.6%) and mitochondria (24.5%) did not change significantly with starvation. There was, however, a decrease in stored lipid (10.7% to 3.2%) and an alteration in the structure of the cristae in mitochondria from red muscle.Atrophy of white muscle fibres is associated with a decrease in both the diameter and fractional volume occupied by myofibrils (85.7% to 61.9% P < 0.01). In a high proportion of white fibres peripheral degeneration of Z-discs is evident causing an unravelling of the thin filament lattice. It is suggested that this allows a partial decrease in myofibril diameter and hence the maintenance of contractile function in muscle from starved fish. In severely degenerating white fibres, disorganised thick and thin filaments and numerous multimembrane lysosome-like vesicles are observed.Starvation results in an increase in the average content of mitochondria in white fibres from 2.2 to 6.7% (P<0.01). In fed plaice mitochondria constitute less than 1% of the volume of the white fibre in 43.5% of the fibres. The proportion of white fibres containing more than 6% mitochondria increases from 6.5% to 58% with starvation.     

Ultrastructure of four types of striated muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary Four types of striated muscle fibers with distinctive ultrastructure were defined in the Atlantic hagfish (Myxine glutinosa, L.): white, intermediate, and red fibers of m. parietalis, and red fibers of m. craniovelaris.White fibers are thick, contain very few mitochondria and fat vacuoles, and possess distinct and separate myofibrils with thin Z-disks and distinct M-lines. Intermediate fibers are thinner, possess largely similar myofibrils that often are even better separated due to a higher content of fat vacuoles and especially mitochondria and glycogen granules. Red fibers of m. parietalis contain large amounts of mitochondria, fat vacuoles, and glycogen granules. Their myofibrils possess M-lines, and although branching more, the myofibrils of red fibers conform with a Fibrillenstruktur pattern like those of white and intermediate fibers. Red fibers of m. craniovelaris are very thin, possess many smaller fat vacuoles, and large amounts of mitochondria and glycogen granules. The myofibrils are significantly thinner than in m. parietalis fibers, run as quite independent well separated units, possess thicker Z-disks, and lack M-lines. Large amounts of myosatellite cells are associated with these red fibers.Triads are located near A/I-junctions in all four fiber types and occur irregularly, the density of triads being different in the various fiber types.We are indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and we also wish to thank Dr. Jan K. S. Jansen, Institute of Physiology, University of Oslo, for valuable suggestions during this study.     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

Myosin and actin in melanophore-like variants of goldfish erythrophoroma cells: their intracellular distribution and possible association with pigment translocation

Summary The occurrence and intracellular distribution of myosin and actin in melanophore-like cells derived from a goldfish erythrophoroma cell line have been studied by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunofluorescence using antisera against chick gizzard myosin heavy chain and carp skeletal muscle actin. SDS-PAGE of the cell extracts separates out one band at 200 kDalton; this is conjugated with the anti-myosin antiserum. Immunofluorescence using the anti-myosin antiserum discloses that myosin in these cells occurs in two forms: discrete, minute clusters and thin filaments bearing a resemblance to stress fibers. The former is distributed evenly over the entire cytoplasm in the cells with dispersed pigments and, upon pigment aggregation, accumulates densely around collapsed melanosomes. The latter runs as thin bundles either radially along the cell center-to-periphery axis or connecting the corners of cell margins; it gives a similar profile in all states of the motile response. Immunofluorescence using the antiactin antiserum or rhodamine-conjugated phalloidin discloses that actin is similarly distributed to myosin, suggesting its possible existence as actomyosin. Simultaneous translocation of the amorphous forms of myosin and actin with melanosomes indicates that they may be involved in pigment migration.     

The cardiac ultrastructure of Chimaera monstrosa L. (Elasmobranchii: Holocephali)

Summary The ultrastructure of the heart in Chimaera monstrosa L. is described. The endocardial and the epicardial cells are similar in the three cardiac regions. Myocardial cells show small variations.The myofibre, 4–6 m thick, contains one or a few myofibrils. Each myosin filament is surrounded by six actin filaments. The sarcomere banding pattern includes the Z-, A-, I-, M-, N-, and H-band. End-to-end attachments between myofibres are composed of alternating desmosomes and fasciae adhaerentes. Desmosomes and nexuses occur between longitudinally oriented cell surfaces. The sarcoplasmic reticulum is poorly developed but well defined. Peripheral coupling-like structures are common, T-tubules are absent. Membrane bound dense bodies occur in all regions. Areas with ribosomes and single myosin filaments are often seen.The epicardial cells have a regular hexagonal surface and are much thicker than the endocardial cells. Numerous short and a few longer cytoplasmic extensions face the pericardial cavity.The fiat endocardial cells contain a large nucleus and small amounts of cytoplasm.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Polarized distribution of γ interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells)

Summary The fine structure of single identified muscle fibers and their nerve terminals in the limb closer muscle of the shore crab Eriphia spinifrons was examined, using a previous classification based on histochemical evidence which recognizes a slow (Type-I) fiber and three fast (Type-II, Type-III, Type-IV) fibers. All four fiber types have a fine structure characteristic of crustacean slow muscle, with 10–12 thin filaments surrounding each thick filament and sarcomere lengths of 6–13 m. Type-IV fibers have sarcomere lengths of 6 m while the other three types have substantially longer sarcomeres (10–13 m). Structural features of nerve terminals revealed excitatory innervation in all four fiber types but inhibitory innervation in Type-I, Type-II, and Type-III fibers only. Thus fibers with longer sarcomeres receive the inhibitor axon but those with shorter sarcomeres do not. Amongst the former, synaptic contact from an inhibitory nerve terminal onto an excitatory one, denoting presynaptic inhibition, was seen in Type-I and Type-II fibers but not in Type-III and Type-IV fibers. Inhibitory innervation of the walking leg closer muscle is therefore highly differentiated: some fibers lack inhibitory nerve terminals, some possess postsynaptic inhibition, and some possess both postsynaptic and presynaptic inhibition.