T-system formation in cultured rat skeletal tissue

By using a lanthanum-staining technique which enhances the visualization of the plasma membrane and its derivatives we have studied the formation of the T system in rat muscle cells differentiating in vitro. We have found that: (1) T-system formation normally occurs after myoblast fusion and is especially extensive in mature myotubes; myoblasts grown in calcium-deficient medium to prevent fusion show increased number of sarcolemmal caveolae but rare, short T tubules. (2) T-system formation in vitro differs from that displayed by rat muscle cells in vivo in that it precedes and is independent of junctional SR differentiation; the uncoordinated development of T tubules and junctional SR in vitro leads to the formation of ‘inverted’ triads and labyrinthine T-system networks. (3) Coated vesicles are frequently found either free in the cytoplasm or associated with growing T tubules in rat muscle cells differentiating in vitro. A role of coated vesicles in T-system formation is proposed.     

SARCOPLASMIC RETICULUM AND T TUBULES IN DIFFERENTIATING RAT SKELETAL MUSCLE

An electron microscope study has been made of the distribution of membrane couplings between the sarcoplasmic reticulum (SR) and either the plasmalemma or the T tubules in fetal and neonatal rat intercostal muscle. Within primitive muscle cells at 12 days of gestation, the SR forms both simple and specialized membrane junctions with the plasmalemma; caveolae are very few, and T tubules are not detected. Undifferentiated cells neighbor muscle cells. Occasionally these cells contain subsurface couplings between the endoplasmic reticulum and plasmalemmae. Possible relationships between these couplings and the peripheral couplings of muscle cells are discussed. By 15–18 days of gestation, caveolae and beaded T tubules, comparable to those of cultured muscle, develop; T tubules lie along-side myofibrils and are rarely transverse. SR couples both to T tubules and to plasmalemmae during this period. T tubules with lineal profiles appear after further development and their orientation transverse to A–I junctions becomes increasingly evident. Membrane couplings between SR and T tubules also increase in number, whereas the incidence of peripheral coupling declines rapidly Evidence suggests that peripheral couplings are swept into myotubes as caveolae proliferate and T tubules form. SR thus appears to initially couple with the plasmalemma and then to await T tubular growth. This contrasts with the developmental pattern described in cultured chick muscle in which peripheral couplings are not reported and T tubules with diads and triads occur at very primitive stages of muscle differentiation.     

Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro

Summary The formation of the sarcoplasmic reticulum (SR) and the transverse tubular system (T-system) in embryonic chick skeletal muscle cells in vitro was studied by either the critical point drying-physical rupturing or physical rupturing-freeze drying together with rotary shadowing. In these cells, two membranous systems were observed. One was composed of flattened sacs which were either isolated or were connected to each other with slender processes to form mostly longitudinally oriented strands. Initially, these sacs had small granules at their surface and were found mainly under the sarcolemma. Later, they became smooth at their surface, extending throughout the cytoplasm to form irregular and dense networks. At later phases, the networks tended to be disposed at right angle to nascent myofibrils, exhibiting a characteristic honeycomb appearance. From the similarities in thin section images, they were identified as developing SR.The other membranous system were tubules with many enlargements. They were frequently associated with coated vesicles which appeared to take part in the formation, elongation, and anastomosing of developing tubules. These tubules could be impregnated with a tannic acid-glutaraldehyde-potassium ferrocyanide complex and, thus, were identified as T-tubules.Abbreviations CPD critical-point drying - ES exoplasmic surface of the sarcolemma - FD freeze-drying - PR physical rupture - PS protoplasmic surface of the sarcolemma - SR sarcoplasmic reticulum - TAGPF tannic acid-glutaraldehyde-potassium ferrocyanide - T-system transverse tubular system     

FREEZE FRACTURE OF SKELETAL MUSCLE FROM THE TARANTULA SPIDER : Structural Differentiations of Sarcoplasmic Reticulum and Transverse Tubular System Membranes

The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.     

Biogenesis of transverse tubules in skeletal muscle in vitro

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.     

The ultrastructure of the striated muscle of the tail of Cercaria chackai Nadakal et al., 1969

The electron microscopic study of the tail of Cercaria chackai reveals that it contains four sets of striated muscle bundles located central to the nonstriated circular and longitudinal muscles. The striated muscle consists of longitudinally oriented lamellar myofibres. Each myofibre contains a single "U" shaped myofibril. The banding pattern is analogous to that of vertebrate striated muscle. The sarcolemma is a simple surface membrane. There are no transverse tubular extensions of sarcolemma. The sarcoplasmic reticulum (SR) is very well developed with cisternae, tubules, and vesicles. SR cisternae form dyadic couplings with the sarcolemma. There is a set of flattened tubules of SR origin traversing the myofibril exactly at the Z region. These tubules are unique to the striated muscle of the cercarian tail and may have functional significance. A diagrammatic reconstruction of the myofibre is presented.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-matched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle.     

A role for phospholipase D3 in myotube formation

Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Summary Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-mateched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle. This research was supported by a grant from the Muscular Dystrophy Association of Canada.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FUSION OF CHICK MYOBLASTS IN VITRO

The process of myoblast fusion was observed in embryonic chick skeletal muscle cells grown in monolayer cultures at the fine structural level. At the first step, the sarcolemmas of cells destined to fuse are closely applied to each other. They are linked in some places by fasciae adherentes; in other places, engulfment of small processes of one cell by another is seen. At a somewhat more advanced stage of myogenesis, vesicles and tubules are formed between the adjacent cytoplasms; presumably, the apposed membranes have opened at several points and their edges have fused to each other. Finally, remnants of cell membranes (vesicles and tubules) disappear completely, and the confluent cytoplasm is formed. The cytoplasmic contents of the multinucleated cells are often poorly admixed, giving the cytoplasm a mosaic appearance in which different zones can be designated as arising from separate cells. This observation suggests, however, that there is slow diffusion of myoblast contents (ribosomes and, possibly, other materials) into the myotube. In agreement with the previous works at the light microscopic level, the present study suggests the occurence of fusion between mononucleated cells, between mononucleated cells and multinucleated myotubes, and between nascent multinucleated myotubes.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

STUDIES OF THE TRIAD : I. Structure of the Junction in Frog Twitch Fibers

The structure of the junction between sarcoplasmic reticulum (SR) and transverse tubular (T) system at the triad has been studied in twitch fibers of the frog. The junction is formed by flattened surfaces of the SR lateral sacs and the T-system tubule, which face each other at a distance of 120–140 A. At periodic intervals of about 300 A, the SR membrane forms small projections, whose tips are joined to the T system membrane by some amorphous material. The SR projections and the amorphous material are here called SR feet. The feet are disposed in two parallel rows, two such rows being present on either side of the T-system tubule. The junctional area between the feet is apparently empty. The feet cover no more than 30% of the T system surface area and 3% of the total SR area. The functional significance of this interpretation of the junctional structure is discussed.     

An ultrastructural study of the body wall muscles of the pentastomid Reighardia sternae dies. 1864 parasitic on the herring gull Larus argentatus pontopp

Summary The fine structure of the body wall muscle of the pentastomid Reighardia sternae is described. The muscle fibres are separated from one another and form two layers, circular and longitudinal. They are cross-striated with approximately 11 actin filaments surrounding each myosin filament. The T-system consists of simple in-pushings of the sarcolemma. The SR is also simple and forms both dyadic and triadic contacts with the T-system tubules and dyadic contacts with the sarcolemma. Electron-dense inclusions occur, usually in the vicinity of the Z-lines, and it is suggested that these may be composed of unsaturated lipids.     

Evidence for a structural relationship between successive parallel tubules in the SR network and supernumerary striations of Z line material in purkinje fibers of the chicken, sheep, dog and rhesus monkey heart.

The striations and the intervening filaments observed in the present study have been variously designated in the literature as: prodomal pattern, leptomeric myofibril, microladder, leptomeric organelle, leptofibril and zebra body. Electron microscope examinations of Purkinje fibers from the septa, papillaries, trabeculae carneae and small endocardial strands from chicken, sheep, dog and monkey hearts have revealed a close association between densely stained striations of supernumerary Z line material and successive parallel tubules in the network formed by the sarcoplasmic reticulum (SR). The striations appear to be linked together by filaments that somewhat resemble the part of thin filaments attached to Z lines in normal fibrils. The evidence for a close association of striations and SR tubules is derived from a similarity of spacing between striations and successive parallel tubules in the SR network and from a resemblance of striation and SR network patterns. The evidence for a structural relationship between striations and SR tubules is derived from the observation of electron-opaque strands traversing the space between striations and SR tubules.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.     

The membrane systems of the cardiac muscle cell of Meganychtiphanes norvegica (M. Sars), euphauciacea,crustacea

Summary The membrane systems of cardiac muscle cells of the euphausiacean Meganychtiphanes norvegica are described. Transverse tubules are found both at the Z-band level (T_z-tubules) and at the H-band level (T_h-tubules). Within the sarcomere narrow longitudinal tubules branch off from the T_z-tubules. At the H-band level these tubules expand forming flattened cisternae in dyadic and triadic couplings with the sarcoplasmic reticulum (SR). Adjacent myofibrils are separated by a well developed SR. Modifications of the SR are seen at the H-band level where junctional cisternae are formed.     

粘虫蛾飞行肌的发育：超微结构特征分析

应用电子显微镜对正常条件下饲养的0-16日龄粘虫Mythimna separata (Walker)雌蛾飞行肌超微结构的研究结果表明：肌原纤维直径。线粒体和横管的体积分量均随蛾龄的增加而增加,到7日龄达到最大值以后又随蛾龄的增加而下降;但是,肌节长度则随蛾龄的增加而缩短,到7日龄达到最短后又随蛾龄的增加而延长,从而使整条发育曲线呈“V”字形;肌原纤维和肌质网体积分量变化不大或无规律可寻;二位体在发育初期和未期的比例较高,而三位体在4和7口龄比例较高;肌丝排列从0～7日龄均是有序的,肌动蛋白丝(细丝)和肌球蛋白丝(粗丝)的数量比为3：1,粗丝的数量变化也不大,每根肌原纤维约有600根。但从10日龄开始肌丝排列出现紊乱,细丝全部消失,粗丝降解、数量减少了30%,从而使肌原纤维留下一片片的空白。根据这些结果,把7日龄前的飞行肌发育过程视为生长过程,而把10日龄后的视作降解过程。最后对粘虫蛾飞行肌与卵巢发育及其飞行能力变化的关系进行了讨论。     

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。