Ultrastructure of the radula protractor of Busycon canaliculatum

Summary The distribution of the sarcoplasmic reticulum and sarcolemmic tubules in the radula protractor muscle of the whelk, Busycon canaliculatum, has been investigated. The sarcoplasmic reticulum consists of an interconnected system of cisternae and tubular channels. The cisternae are closely associated with the sarcolemma. The tubular channels project from the cisternae into the interior of the cell and run parallel to the long axis of the myofilaments. Parallel tubular channels are interconnected with one another by short branches. This finding of an elaborate sarcoplasmic reticulum supports previous physiological work on this smooth muscle which indicated the presence of an intracellular compartmentalization of calcium ions. There is also an extensive system of tubular invaginations of the sarcolemma which we have termed sarcolemmic tubules. These tubules are 600 Å in diameter and about 0.5 microns in length. There is a substructure associated with the leaflet of the tubular membrane bordering the extracellular space. The sarcolemmic tubules penetrate only half a micron from the surface of the cell and interdigitate with the sarcoplasmic reticulum associated with the sarcolemma. Calculations have shown that the surface area of this smooth muscle cell is more than doubled by the presence of sarcolemmic tubules.     

The relation between excitation-contraction coupling and fine structure of a molluscan muscle,the radular retractor of the whelk,Buccinum undatum

The Buccinum radula is of the rachiglossate type with two outer rows of fierce hook-like attack teeth and a medial row of straight sharp-pointed shredding teeth. Individual cells of the radular retractor muscle are 10–12 m in diameter and separated at the closest by gaps of only 40 nm, providing areas of potential electrical contact. The cell membranes are heavily invested with long finger-like invaginations, associated with sarcoplasmic reticular cisternae, and surface caveolae; the latter are associated with the numerous dense body membrane attachment plaques found in this muscle. The radular retractor muscle possesses a significant sarcoplasmic reticulum of peripheral cisternae and deeper vesicles associated with mitochondria. The surface caveolae may result from myofilament force exerted via attachment plaques at the cell membrane, while deeper invaginations may constitute a rudimentary transverse tubular system to relay surface depolarization to associated sarcoplasmic reticular cisternae inducing calcium release to effect excitation-contraction coupling. The radular retractor muscle possesses the usual thick paramyosin and thin actin myofilaments, the latter associated with dense bodies and attachment plaques presumably to transduce force to the cell membrane. The mitochondria are unusually large and packed into dense central clusters surrounded by large deposits of glycogen granules. The nerve endings on the radular retractor muscle fibres show four different types of transmitter vesicle, presumably related to the four kinds of agonist action in this muscle, cholinergic, serotonergic, peptidergic and purinergic. All nerve endings have mixed vesicle populations, clear evidence of co-transmission. In this muscle we see a modification of usual smooth muscle structure to effect fast sustained contractions, an ultrastructural configuration functionally designed for the muscle's central role in the feeding cycle.Abbreviations ABRM anterior byssus retractor muscle - EC coupling excitation-contraction coupling - RP radular protractor muscle - RR radular retractor muscle - SR sarcoplasmic reticulum - T-system transverse tubular system     

THE FINE STRUCTURE OF NEUROMUSCULAR JUNCTIONS AND THE SARCOPLASMIC RETICULUM OF EXTRINSIC EYE MUSCLES OF FUNDULUS HETEROCLITUS

The extrinsic eye muscles of the killifish (F. heteroclitus) were fixed in OSO₄ (pH 7.6) and subsequently dehydrated, embedded, and sectioned for electron microscopy. The fine structures of neuromuscular junctions and of sarcoplasmic reticulum were then observed. The neuromuscular junction consists of the apposition of axolemma (60 to 70 Å) and sarcolemma (90 to 100 Å), with an intervening cleft space of 200 to 300 Å, forming a synaptolemma 400 to 500 Å thick. The terminal axons contain synaptic vesicles, mitochondria, and agranular reticulum. The subsynaptic sarcolemma lacks the infolding arrangement characteristic of neuromuscular junctions from other vertebrate skeletal muscle, making them more nearly like that of insect neuromuscular junctions. A comparison between the folded and non-folded subsynaptic membrane types is made and discussed in terms of comparative rates of acetylcholine diffusion from the synaptic cleft and resistances of the clefts and subsynaptic membranes. The sarcoplasmic reticulum consists of segmentally arranged, membrane-limited vesicles and tubular and cisternal elements which surround individual myofibrils in a sleeve-like arrangement. Triadic differentiation occurs at or near the A-I junction. Unit sleeves span the A and I bands alternately and consist of closed terminal cisternae interconnected across the A and I bands by tubular cisternae. The thickness of the sarcoplasmic membranes increases from 30 to 40 Å in intertriadic regions to 50 to 70 Å at the triads. The location of the triads is compared with previously described striated muscle from Ambystoma larval myotomes, cardiac and sartorius muscles of the albino rat, mouse limb muscle, chameleon lizard muscle, and insect muscle, with reference to their possible role in intracellular impulse conduction.     

On the Organisation of the Sarcotubular Systems in the Caudal Muscle Cells of Larvaceans (Tunicata)

The structure of the sarcotubular systems of the caudal muscle cells is described in various larvaceans. In Oikopleura there is both a transverse tubular system and a sarcoplasmic reticulum; there are internal couplings between the two and also sarcolemmal couplings. In Fritillaria (and probably also in Kowalevskaia), a transverse tubular system is lacking, and there are only sarcolemmal couplings with the sarcoplasmic reticulum, which is related to the mitochondria as well as to the myofilaments. The significance of these differences is discussed, and it is concluded that the arrangement of the sarcotubular systems is related to muscle fibre thickness; within the Tunicata, these systems do not indicate phylogenetic relationships.     

On the Presence of a Transverse System in Tunicate Muscle

Abstract Ultrastructural investigation of the caudal muscle cells in the tadpole larva of the ascidian Dendrodoa grossularia has shown that in this species, a transverse tubular system is present; diad and triad couplings are formed with the sarcoplasmic reticulum. Peripheral couplings of the sarcoplasmic reticulum are infrequent and have only been observed at the longitudinally apposed faces of the caudal muscle cells.     

Larval convergence in a colonial tunicate: the organization of the sarcotubular complex in <Emphasis Type="Italic">Ecteinascidia turbinata</Emphasis> (Perophoridae,Phlebobranchiata, Tunicata,Chordata)

Ecteinascidia turbinata is a colonial ascidian that as an adult shares characters with phlebobranch ascidians, whereas the larvae are similar to aplousobranch ascidian larvae. The sarcotubular complex consists of invaginations of the sarcolemma that contact the sarcoplasmatic reticulum via dyads or triads. If present, the invaginations of the sarcolemma in tunicates have been characterized as laminar or tubular. We comparatively investigated the sarcotubular complex of E. turbinata and seven other tunicate species using 3D-reconstruction techniques based on electron micrographs of serial sections. The mononucleate muscle cells in E. turbinata possess intermediate and close junctions and contain several layers of peripheral myofibrillae. The myofibrillae are surrounded by continuous cisternae of the sarcoplasmic reticulum that forms interconnected rings around the z-bands. The invaginations of the sarcolemma are laminar, contacting the sarcoplasamatic reticulum at the height of the z-bands via dyads and triads. We present a clear definition of character states encountered in Tunicata: laminar invaginations are characterized by a width to length ratio of smaller than 1:20, tubular invagination by a width to length ratio of larger than 1:10. Laminar invaginations are found in stolidobranch ascidians and E. turbinata. Tubular invaginations are present in aplousobranch ascidians and appendicularians. This character state distribution across taxa supports the hypothesis that E. turbinata should be included in Phlebobranchiata as suggested by adult characters and that the larval similarities with Aplousobranchiata arose by convergent evolution. An erratum to this article can be found at     

A transmission delay and the effect of temperature at the triadic junction of skeletal muscle

The coupling process at the triadic junctions in skeletal muscle fibres is characterized by a significant latency between the depolarization of the transverse tubular membrane and the release of Ca from the sarcoplasmic reticulum. This time interval, the triadic delay, is sufficiently long to allow for the participation of a chemical process. The strong temperature dependence of the triadic delay (Q10 near 2.7) suggests that a sequence of chemical steps may link the electrical signal in the T-tubules to the opening of Ca channels in the terminal cisternae of the sarcoplasmic reticulum.     

THE ORGANIZATION OF THE FLIGHT MUSCLE IN A DRAGONFLY, AESHNA SP. (ODONATA)

The structure of the flight muscle of a dragonfly (Aeshna sp.) has been studied with the light and electron microscopes, and the organization of this specialized tubular muscle is described. This tissue is characterized by the great development of the sarcosomes, which are slab-like and are arranged within the fiber opposite each sarcomere of the radially oriented lamellar myofibrils. A well developed and highly ordered sarcoplasmic reticulum is present, consisting of perforated curtain-like cisternae extending across the face of each fibril, together with tubular invaginations of the fiber plasma membrane situated within indentations in the sarcosomes and traversing the fibril surface midway between the Z and M levels. The structure of these fibers, and notably the organization of the reticulum, is compared with that of other types of muscle, and the possible role of the two components of the sarcoplasmic reticulum in the contraction physiology of the dragonfly muscle fiber is discussed.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparative study of the organization of the sarcotubular system in ascidian muscle

The caudal musculature of ascidian tadpole larvae consists of mononucleated muscle cells joined end to end in long rows flanking the notochord. A comparative study of the fine structure of these cells in larvae from different families has revealed wide variations in the pattern of organization of the sarcotubular system. The species examined can be distinguished in two groups according to the presence or absence of a system of plasma membrane invaginations equivalent to the T system of vertebrate and invertebrate striated muscle. Muscle cells from the first group of species, Clavelina lepadiformis, Ciona intestinalis and Molgula socialis, are characterized by absence of T system and show peripheral couplings of sarcoplasmic reticulum cisternae directly with the plasma membrane. In contrast, a T system is present in muscle cells of Diplosoma listerianum, Styela plicata and Botrylloides leachi. The presence of T system in ascidian muscle is not related to the taxonomic position of the various species, but rather to the intracellular disposition of the myofibrils, which are peripheral in the species of the first group whereas they occupy a more internal position in the species of the second group. The T system displays unique structural features in ascidian muscle. It consists of wide laminae invaginating from the plasma membrane and associated in longitudinally oriented dyads with sarcoplasmic reticulum cisternae in register with the I band of the myofibrils. It is apparent from these observations that, in contrast with the uniformity of myofibrillar structure in all chordates, there are basic differences between ascidians and vertebrates as regards the organization of the sarcotubular system. On the other hand, there are significant similarities in this respect between ascidian and invertebrate muscle.     

Specialized contacts between sarcolemma and sarcoplasmic reticulum at the ends of muscle fibers in the diaphragm of the rat

Summary An electron-microscopic study of the myotendinous portion of the diaphragm in the Wistar rat has shown that at the ends of muscle fibers, longitudinally oriented invaginations and peripheral furrows of the sarcolemma establish specialized contacts with individual sacs of the sarcoplasmic reticulum. The construction of these terminal contacts is similar to that of contacts between sarcolemmic T-tubules and terminal cisternae of the sarcoplasmic reticulum, characterized by formation of triads. The contact zones of the sac membrane are undulated and bound to the adjoining sarcolemma via electron-dense profiles of varying forms. Frequently, the terminal contacts and triads are located at the same level within the muscle fiber, at the borderline between A- and I-bands of the sarcomeres. At the ends of muscle fibers combined contacts between peripheral furrows of the sarcolemma, terminal cisternae of the sarcoplasmic reticulum, and T-tubules of the triads are also disclosed. The implications of the terminal contacts for muscle contraction are discussed.     

A STRAND OF CARDIAC MUSCLE : Its Ultrastructure and the Electrophysiological Implications of Its Geometry

The structure of a small strand of rabbit heart muscle fibers (trabecula carnea), 30–80 µ in diameter, has been examined with light and electron microscopy. By establishing a correlation between the appearance of regions of close fiber contact in light and electron microscopy, the extent and distribution of regions of close apposition of fibers has been evaluated in approximately 200 µ length of a strand. The distribution of possible regions of resistive coupling between fibers has been approximated by a model system of cables. The theoretical linear electrical properties of such a system have been analyzed and the implications of the results of this analysis are discussed. Since this preparation is to be used for correlated studies of the electrical, mechanical, and cytochemical properties of cardiac muscle, a comprehensive study of the morphology of this preparation has been made. The muscle fibers in it are distinguished from those of the rabbit papillary muscle, in that they have no triads and have a kind of mitochondrion not found in papillary muscle. No evidence of a transverse tubular system was found, but junctions of cisternae of the sarcoplasmic reticulum and the sarcolemma, peripheral couplings, were present. The electrophysiological implications of the absence of transverse tubules are discussed. The cisternae of the couplings showed periodic tubular extensions toward the sarcolemma. A regularly spaced array of Z line-like material was observed, suggesting a possible mechanism for sarcomere growth.     

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.     

THE SARCOPLASMIC RETICULUM, THE T SYSTEM, AND THE MOTOR TERMINALS OF SLOW AND TWITCH MUSCLE FIBERS IN THE GARTER SNAKE

Twitch and slow muscle fibers, identified morphologically in the garter snake, have been examined in the electron microscope. The transverse tubular system and the sarcoplasmic reticulum are separate entities distinct from each other. In twitch fibers, the tubular system and the dilated sacs of the sarcoplasmic reticulum form triads at the level of junction of A and I bands. In the slow fibers, the sarcoplasmic reticulum is severely depleted in amount and the transverse tubular system is completely absent. The junctional folds of the postsynaptic membrane of the muscle fiber under an "en grappe" ending of a slow fiber are not so frequent or regular in occurrence or so wide or so long as under the "en plaque" ending of a twitch fiber. Some physiological implications of these differences in fine structure of twitch and slow fibers are discussed. The absence of the transverse tubular system and reduction in amount of sarcoplasmic reticulum, along with the consequent disposition of the fibrils, the occurrence of multiple nerve terminals, and the degree of complexity of the post junctional folds of the sarcolemma appear to be the morphological basis for the physiological reaction of slow muscle fibers.     

ELECTROMECHANICAL COUPLING IN TUBULAR MUSCLE FIBERS : I. The Organization of Tubular Muscle Fibers in the Scorpion Leiurus quinquestriatus

The tubular fibers of the claw-closer muscle of the scorpion have a central core containing nuclei and mitochondria. The myofibrils have the shape of thin lamellae (1 µ) extending radially from the core to the surface membrane (20 µ). The thick myofilaments are organized in a hexagonal array with orbits of 10–13 thin myofilaments. The ratio of thick-to-thin filaments is 1:5. Transverse tubular system (TS) openings are located between lamellated myofibrils. In each sarcomere two TS's are found, one on each side of the H band. The TS is composed of a transverse tubule and tubular pockets (TP). The TP's form diadic contact with the terminal cisternae of the sarcoplasmic reticulum. The TS can be traced from the cell membrane down to the cell core. The surface area of the TS was calculated to be six times that of the outer surface membrane.     

The fine structure of muscle in a marine turbellarian

Summary The fine structure of the myofibers of Notoplana acticola as studied by electron microscopy indicates that they are composed of thick myofilaments about 200 Å wide with tapering ends and thin myofilaments about 50 Å wide, arranged alongside each other parallel to the long axis of the cell. There is no orderly transverse arrangement of filaments; instead they appear staggered in the fiber. In cross sections 6 to 10 thin filaments form an orbit around one thick filament with possible cross-linkage between the two types of filaments.Dense bodies are associated with the sarcolemma and with the sarcoplasmic reticulum, and appear to serve as attachments for the thin filaments. Dense bodies are compared to elements forming a fragmented Z-disc.Mitochondria, situated in the periphery or the center of fibers, are associated with granules interpreted as glycogen.The sarcoplasmic reticulum consists of: sacs or cisternae in close proximity to the sarcolemma, longitudinal tubular elements between and parallel to the myofilaments, and a tubular network around the filaments. There is no well-defined sarcolemmal-derived transverse tubule system as described in striated muscles. It is hypothesized that in these muscles, the functional equivalent of the T system may be the area of sarcolemma in contact with the cisternae of the sarcoplasmic reticulum.This work was supported by Grant No. GM 10292 from the U. S. Public Health Service to Professor Richard M. Eakin, Department of Zoology at the University of California, Berkeley, USA, where this investigation was conducted during the author's sabbatical leave of absence from the University of Illinois.I wish to thank Professor Eakin for valuable discussions and for his kind hospitality in extending the facilities of his laboratory and the use of the electron microscope to me, and the John Simon Guggenheim Memorial Foundation for the Fellowship which I held during 1964–65.     

Joint participation of mitochondria and sarcoplasmic reticulum in the formation of tubular aggregates in gastrocnemius muscle of CK-/- mice

Tubular aggregates are specific subcellular structures that appear in skeletal muscle fibres under different pathological conditions. The origin of the tubular aggregates is generally ascribed to proliferating membranes of sarcoplasmic reticulum. There are, however, histochemical indications for the presence of mitochondrial enzymes in tubular aggregates suggesting contribution of mitochondria to the genesis of tubular aggregates. In this study we used an immunocytochemical detection technique to assess participation of mitochondria and of sarcoplasmic reticulum in derivation of tubular aggregates. The fast skeletal muscle fibres (m. gastrocnemius) of mice bearing the double invalidation for both the mitochondrial and the cytosolic isoforms of creatine kinase (CK), an enzyme involved in energetics of muscle cells, were employed as a model muscle with tubular aggregates (Steeghs et al., Cell 89, 93-103, 1997). Immunogold labelling of the bc1 complex, a specific integral protein of the inner mitochondrial membrane, provided strong signals in both the mitochondria and tubular aggregates but not in other ultrastructural components of muscle fibres. A similar strong immunogold signal was obtained when labelling for SERCA1, a specific enzyme of the sarcoplasmic reticulum membrane, in regions of typical occurrence of the sarcoplasmic reticulum and in tubular aggregates. In double labelling experiments, we found simultaneous labelling of tubular aggregates with both the bc1 and SERCA1 antibodies. It is concluded, that in CK-/- mouse both the inner mitochondrial membrane and the membrane of the sarcoplasmic reticulum participate in the formation of tubular aggregates.     

On the unity of cytomembrane system in the skeletal muscle

In situ cytochemical evidence for specific Ca-binding sites in the cytomembrane system of skeletal muscle fibers is reported. High Ca accumulation was found at the junctions between different types of cytomembranes. Such junctions might represent "gate-locks' for intracellular Ca movements. Openings of sarcoplasmic reticulum (SR) in frog muscle fibers and of T-tubules in rat muscle fibers are described. Coated and noncoated caveolae were found in rat muscle fibers. The same positive reaction for TPP-ase was found in trans-Golgi zone, terminal cisternae and subsarcolemmal cisternae. These results suggest the membrane continuity and ontogenetic relationships in the cytomembrane system of skeletal muscle fibers.     

THE SARCOPLASMIC RETICULUM AND ITS ASSOCIATION WITH THE T SYSTEM IN AN INSECT

The fine structure of the sarcoplasmic reticulum and the transverse tubular system of the femoral muscle of the cockroach, Leucophaea maderae, was studied after prefixation in glutaraldehyde, postfixation in osmium tetroxide, and embedding in Epon. The sarcoplasmic reticulum in this muscle reveals features not previously reported. The sarcoplasmic reticulum is abundant, consisting mainly of a fenestrated envelope which surrounds each myofibril at all levels in the sarcomere. This sarcoplasmic reticulum envelope is continuous transversally as well as longitudinally along the myofibrils. Dyadic junctions are formed by a single T system element which contacts the unfenestrated sarcoplasmic reticulum of adjacent myofibrils in an alternating manner at the ends of the A band. At the dyads, regularly spaced thickenings of the sarcoplasmic reticulum membranes bordering the dyadic spaces are noted. These thickenings, however, do not contact the T tubule membrane. Typical dyadic contacts also are seen between the cell surface membrane and sarcoplasmic reticulum. Z line-like material is seen in contact with the membranes of the cell surface and longitudinal branches of the T systems.