MODELS OF MUSCLE Z-BAND FINE STRUCTURE BASED ON A LOOPING FILAMENT CONFIGURATION

The fine architecture of skeletal muscle Z bands is considered in view of stereo electron microscopical evidence and current biochemical and immunological concepts, and a new Z-band model is proposed. This model is based on a looping, interlinking configuration, within the Z band, of strands which emanate from I-band (actin) filaments of adjacent sarcomeres. Two versions of the model seem presently feasible: one in which the Z-band lattice is composed of actin loops; and another in which the same pattern is derived from tropomyosin. Either version satisfies actual electron micrograph images as well as or better than prior Z-band models. Moreover, the principle of looping linkage in filament-to-filament attachment can be related to similar filament patterns seen in several adhesion sites where intracellular filaments insert on cell membranes.     

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.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Three-dimensional reconstruction of a simple Z-band in fish muscle

The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.     

Symmetry of a Vertebrate Muscle Basketweave Z-Band

The Z-band in vertebrate striated muscle links actin filaments of opposite polarity in adjacent sarcomeres to form a regular structure based on a tetragonal lattice. In transverse sections there are two commonly observed appearances of the Z-band seen in different muscles, namely, the small-square lattice and the basketweave forms. A clear example of the latter occurs in the fin muscle of the flatfish plaice and its symmetry is described here. Improved methods over previous work include fast freezing/freeze-substitution and lattice straightening of the scanned images. It is demonstrated here that when a longitudinal section is tilted in the electron microscope about the myofibril axis, the 10 and 01 projections are mirror images of each other about the centre of the Z-hand. By examining the symmetry relationships between these views and a longitudinal 11 projection and a transverse view, it is concluded that the symmetry is best described by the two-sided plane group c12. The twofold axis lies in the central plane of the Z-band along the diagonal of the primitive lattice and runs normal to the actin filaments. In contrast, the symmetry of the simple Z-band in fish myotomal white muscle, which in longitudinal sections has the appearance of a single zigzag structure, is p12₁ (Luther, P. K. (1991), J. Cell Biol. 113, 1043-1055).     

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.     

粘虫蛾飞行肌超微结构的研究

应用电子显微镜对粘虫雌蛾Mythimna separata(Walker)飞行(背纵)肌的研究结果表明,其肌原纤维由500-700根肌球蛋白丝(粗丝)组成,每根粗丝由6根肌动蛋白丝(细丝)环绕排列成六角形,每根细丝精确地位于两根粗丝间1/2处,从而使粗丝和细丝的比为1：3。肌节较短,长度约2.2-2.6μm。肌原纤维之间充满着线粒体和横管。每个肌节约有线粒体三个,横管二根。线粒体约占肌纤维体积的40%,而横管为7%。每根横管准确地位于肌节的1/4、3/4处,或Z线和中膈的中央,并与肌质网交接形成二位体(dyads)或三位体(triads)。肌质网相当不发达,约占肌纤维体积的2.5%。但其分布很有特色,即除了紧贴于肌原纤维周围的由单层液泡组成的肌质网以外,在中膈处还有一层横穿于肌原纤维的肌质网。和其它同步飞行肌的结构和功能分析比较的结果还表明,粘虫蛾飞行肌具有较善于飞行的结构。     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

FILAMENT ULTRASTRUCTURE AND ORGANIZATION IN VERTEBRATE SMOOTH MUSCLE : Contraction Hypothesis Based on Localization of Actin and Myosin

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15–25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.     

Three-dimensional reconstruction of the Z disk of sectioned bee flight muscle

The three-dimensional structure of the central region of the Z disk of honeybee flight muscle has been determined to a resolution of 70 A by three-dimensional reconstruction from electron micrographs of tilted thin sections. The reconstructions show a complex assembly in which actin filaments terminate and are cross-linked together; a number of structural domains of this network are resolved in quantitative three-dimensional detail. The central region of the Z disk contains two sets of overlapping actin filaments of opposite polarity, which originate in the sarcomeres adjacent to the Z disk, and connections between these filaments. The filaments are deflected by the attachment of cross-links; spacing between filaments change by greater than 100 A during their passage through the Z disk. Each actin filament is linked by connecting structures to four filaments of opposite polarity and two filaments are of the same polarity. Four types of connecting density domain are observed in association with pairs of filaments of opposite polarity: C1, C2, C3, and C5. Two of these, C3 and C5, are associated with the ends of actin filaments. Another connection, C4, is associated with three filaments of the same polarity; C4 is threefold symmetric.     

Z-line formins promote contractile lattice growth and maintenance in striated muscles of C. elegans

Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line-associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.     

THE STRUCTURE OF A SIMPLE Z LINE

The structural simplicity of the Z line in fish muscle fibers allows direct visualization of its basic geometry. Models which postulate termination of the I filaments at the edges of the Z line and the direct linkage of I filaments belonging to the two adjacent sarcomeres by Z filaments crossing the whole width of the Z line give the best fit to the electron micrographs. The structure of fish Z lines is not significantly altered by the use of different fixation procedures and by changes in sarcomere length.     

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.     

The existence of an insoluble Z disc scaffold in chicken skeletal muscle

Extraction of glycerinated chicken skeletal muscle with 0.6 M potassium iodide leaves a framework of insoluble components within each muscle fiber. This framework is composed primarily of planes of in-register Z discs that have been thickened by the accumulation of material on both sides of each disc during extraction. Membrane vesicles, presumably remnants of the T system, remain surrounding the Z discs. When the framework is sheared in a blender, it is preferentially cleaved between Z planes, resulting in the formation of large sheets of interconnected, closely packed Z discs in a honeycomb-like array. Cleavage occurs in regions formerly occupied by the A bands, which have been weakened by the removal of myosin. The existence and stability of these planar Z disc arrays demonstrate the presence and strength of connections between adjacent myofibrils.SDS-polyacrylamide gel electrophoresis reveals that this framework consists primarily of actin and desmin, with lesser amounts of a few proteins including α-actinin, myosin and tropomyosin. Z disc sheets and KI-extracted myofibrils provide a distinct face-on view and side view, respectively, of the Z disc. In indirect immunofluorescence, these two views have revealed that desmin is present at the periphery of each Z disc, forming a network of proteinaceous collars within the Z plane. α-Actinin is localized within each disc, giving a face-on fluorescence pattern that is complementary to that of desmin. Actin is present throughout the thickened Z plane, while myosin and tropomyosin exist only in the insoluble residue that coalesces on both faces of each disc.We conclude that desmin, perhaps in conjunction with actin, is responsible for interlinking Z discs of adjacent myofibrils, and may thus serve as a mechanical and structural integrator of muscle fibers. Its hydrophobic nature and coincident distribution with the T system suggest that it may also be responsible for mediating filament-membrane interactions and anchoring the triad to the Z disc. Its collar-like distribution suggests that it may aid in maintaining the structural integrity of the Z disc and the actin filaments inserted into it.     

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.     

MECHANISM OF SUPERCONTRACTION IN A STRIATED MUSCLE

The phenomenon of contraction of a striated muscle down to below 50 per cent rest length has been examined for the scutal depressor of the barnacle Balanus nubilus by a combination of phase contrast and electron microscopy. It was found that neurally evoked contraction down to 60 per cent rest length results from the shortening of the I band. At the same time the Z disc changes in structure by an active process which results in spaces opening up within it. Thick filaments can now pass through these spaces from adjacent sarcomeres, interdigitating across the discs. Interdigitation permits repetitive contraction in the living muscle to below 30 per cent rest length. In non-neurally evoked contractions most thick filaments do not find spaces in the Z disc and bend back, giving rise to contraction band artifacts. Expansion of the Z disc can be produced in glycerinated material by the addition of solutions containing a high concentration of ATP.     

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.     

Desmin and vimentin coexist at the periphery of the myofibril Z disc.

Two-dimensional gel electrophoresis has revealed that vimentin, the predominant subunit of intermediate filaments in cells of mesenchymal origin, is a component of isolated skeletal myofibrils. It thus coexists in mature muscle fibers with desmin, the major subunit of muscle intermediate filaments. Antisera to desmin and vimentin, shown to be specific for their respective antigens by two-dimensional immunoautoradiography, have been used in immunofluorescence to demonstrate that vimentin has the same distribution as desmin in skeletal muscle. Both desmin and vimentin surround each myofibril Z disc and form honeycomb-like networks within each Z plane of the muscle fiber. This distribution is complementary to that of alpha-actinin within a given Z plane. Desmin and vimentin may thus be involved in maintaining the lateral registration of sarcomeres by transversely linking adjacent myofibrils at their Z discs. This linkage would support and integrate the fiber as a whole, and provide a molecular basis for the cross-striated appearance of skeletal muscle.     

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.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.