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.     

Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)

The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, mid-midgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or double-membraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic.     

Development,cytology, lipid storage and motility of the Malpighian tubules of the nymphal dragonfly,Aeshna cyanea (Müller) (Odonata : Aeshnidae)

The Malpighian tubules of nymphal Aeshna cyanea (Odonata : Aeshnidae) were examined by light and electron microscopy. The 1st-instar nymphs have only 3 branchless tubules. With proceeding nymphal stages, these lengthen and branch. Also, additional tubules bud from the gut and show the same pattern of growth and branching, until in the final instar up to 21 separate tufts of branched tubules are present. A serpentine trachea/tracheole and a cross-striated muscle are helically wound around each tubule in close apposition. Isolated tubules show twisting movements for several days. Contraction of the muscle is responsible for fast coiling movements, while the slow decoiling movements probably depend on elastic deformations of the accompanying trachea, the basal lamina and the tubule cells, the latter showing an elaborate cytoskeleton and multiple adhesive junctions.The tubular epithelium consists of 4 types of cells. The distal segment is composed of ion transporting cells and terminates with a short, solid tip segment of undifferentiated cells. The intermediate segment consists of lipid cells which are densely filled with triglyceride droplets, as revealed by thin layer chromatography. Lipid cells are already present in the 1st instar before the nymphs have taken up any food. In later instars, the renal lipid content varies to some extent with the nutritional state and is nearly depleted during metamorphosis. The proximal segment is the region of tubular branching and may be conceived as the collecting duct of each tuft. Its epithelium consists of mucocytes.     

The ultrastructural differentiation of the clonal myogenic cell line L6 in normal and high K+ medium

Cells of the clonal myogenic line L6 were examined electron microscopically at successive stages of growth. L6 cells are developmentally similar to those of chick and rat primary skeletal muscle cultures and skeletal muscle in vivo, with respect to myofibrillogenesis and sarcomere organization. However, the sarcoplasmic reticulum and T systems of L6 myotubes are not as well differentiated as those of primary muscle cultures and adult skeletal muscle. Finally, L6 myotubes show precocious sarcomere differentiation when cultured in medium containing 25 mM potassium.     

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells

We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K _m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K _m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.     

Ultrastructure and cytochemistry of salivary glands of the predator Podisus nigrispinus (Hemiptera: Pentatomidae)

Podisus nigrispinus Dallas (Hemiptera: Pentatomidae) is a zoophytophagous insect with a potential for use as a biological control agent in agriculture because nymphs and adults actively prey on various insects by inserting mouthparts and regurgitating the contents of the salivary glands inside the prey, causing rapid paralysis and death. However, the substances found in saliva of P. nigrispinus that causes the death of the prey are unknown. As a first step to identify the component of the saliva of P. nigrispinus, this study evaluated the ultrastructure and cytochemistry of the salivary glands of P. nigrispinus. The salivary system of P. nigrispinus has a pair of principal salivary glands, which are bilobed with a short anterior lobe and a long posterior lobe, and a pair of tubular accessory glands. The principal gland epithelium is composed of a single layer of cells enclosing a large lumen. Epithelial cells of the principal salivary gland vary from cubic to columnar shape, with one or two spherical and well-developed nuclei. Cells of the anterior lobe of the principal salivary gland have an apical surface with narrow, short, and irregular plasma membrane foldings; apical and perinuclear cytoplasm rich in rough endoplasmic reticulum; and mitochondria with tubular cristae. The basal portion of the secretory cells has mitochondria associated with many basal plasma membrane infoldings that are short but form large extracellular canals. Secretory granules with electron-dense core and electron-transparent peripheral are dispersed throughout the cytoplasm. Cells of the posterior lobe of the principal salivary gland are similar to those of the anterior lobe, except for the presence of mitochondria with transverse cristae. The accessory salivary gland cells are columnar with apical microvilli, have well-developed nucleus and cytoplasm rich in rough endoplasmic reticulum, and have secretory granules. Cytochemical tests showed positive reactions for carbohydrate, protein, and acid phosphatase in different regions of the glandular system. The principal salivary glands of P. nigrispinus do not have muscle cells attached to its wall, suggesting that saliva-releasing mechanism may occurs with the participation of some thorax muscles. The cytochemical and ultrastructural features suggest that the principal and accessory salivary glands play a role in protein synthesis of the saliva.     

THE ULTRASTRUCTURE OF MAMMALIAN CARDIAC MUSCLE

Papillary muscles of rat and dog hearts were fixed in such a way as to prevent excessive shortening during the procedure. The material was embedded in either araldite or methacrylate and was stained in various ways. The filamentous fine structure of mammalian cardiac muscle is similar to that previously described for striated skeletal muscle. The sarcomeres are composed of a set of thick and thin filaments which interdigitate in the A band proper. The filament ratios and the filamentous array are in accord with those found in skeletal muscle. The functional significance of this twofold array of filaments is not entirely clear. Various other structural aspects of cardiac cells such as surface membranes, mitochondria, nuclei, and cytoplasmic granules are described. The sarcoplasmic reticulum is discussed in detail as are the various structural components forming the intercalated discs. Fairly frequent deep invaginations of the sarcolemma with basement membrane are observed in addition to the intercalated discs. These surface membrane invaginations probably explain the branching appearance of cardiac fibers as seen under the light microscope.     

FORMATION OF ELABORATE NETWORKS OF T-SYSTEM TUBULES IN CULTURED SKELETAL MUSCLE WITH SPECIAL REFERENCE TO THE T-SYSTEM FORMATION

Muscle cells, cultured for 1–28 days from 11-day chick embryo breast muscles, often show elaborate, three-dimensional networks of a membranous system. The network consists of tubular units which are quite regularly arranged. The tubular units composing the network are accessible to ferritin particles suspended in the culture medium; this suggests continuity with the extracellular fluid. These networks can be regarded as a special morphological elaboration of the T-system tubules. Such network formations can be seen much more often in well-developed myotubes. The networks usually exhibit a hexagonal pattern, which is formed of tubular units of a constant diameter. However, some early myotubes contain tetragonal networks, which are composed of spherical pockets with channels of lesser diameter connecting the pockets. Networks are also observed which probably represent a transitional form between these two patterns. Myotubes show many inpocketings of the sarcolemma similar to what are commonly referred to as caveolae or micropinocytotic vesicles. The similarity in configuration and dimension of the tubular units of the network to the caveolae leads to the plausible suggestion that repeated caveola-formation from the sarcolemma or T-system tubule may result in formation of these networks. In this connection, a possible mechanism of the T-system tubule formation is discussed.     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Ultrastructural study of synovial membrane from the antebrachiocarpal joint of calves

The synovial intima from the antebrachiocarpal joint of 4-month-old calves was between 1 and 3 cells in thickness and did not have a basal lamina. Numerous areas of the intimal matrix were in direct contact with the joint lumen. The synovial membrane was comprised mainly of A-type synoviocytes usually located adjacent to the joint lumen. These cells were characterized by numerous filopodia (or lamellipodia), large, empty-appearing vacuoles, numerous lysosomes, large vacuoles containing granular material separated from the vacuolar membrane by a radiolucent band, and coated micropinocytotic vesicles. Smooth micropinocytotic vesicles were seen only rarely in these cells. In contrast, B-type cells had few filopodia, numerous smooth micropinocytotic vesicles, few coated micropinocytotic vesicles, a well-developed Golgi apparatus and rough endoplasmic reticulum, mitochondria that were longer and had a denser matrix than that of A cell mitochondria, and surprisingly, only few maturing or fully formed secretory granules. A distinct intermediate (C or AB) type synoviocyte could not be unequivocally identified. Desmosome-like structures were present between synoviocytes, although it was considered questionable if these were true intercellular junctions. No other junctions were present.     

Regeneration of muscles after cardiotoxin injury. I. Cytological aspects

Regeneration of several adult rat and mouse skeletal muscles was studied after degeneration of muscle fibers had been obtained by the selective action of the cardiotoxin of Naja mossambica mossambica venom. Experimental conditions were set up to ensure minimal damage to satellite cells and also the nerves and blood vessels of the original muscles. As in the other types of experimental regeneration, the structure of the regenerated muscle appeared in many respects different from that of the normal muscle. Moreover the neuromuscular junctions of 'en plaque' type were transformed to 'en grappe' type junctions. Many ultrastructural abnormalities often displayed by these junctions might be linked, at least partially, to the persistence in the regenerating muscle of the original synaptic basal lamina sheaths and their inductive properties.     

Fluctuation in the development of various skeletal muscles in the chick embryo,with special reference to AChE activity and the formation of neuromuscular junctions

Acetylcholinesterase (AChE)-rich cytoplasmic granules in the developing myofibers increased remarkably until the establishment of neuromuscular junctions and thereafter decreased rapidly, whereas junctional AChE activities continued to increase (K. Wake, 1976, Cell Tissue Res. 173, 383–400). In the present paper, during the developmental course of the chick embryo, the temporal and regional gradients in differentiation of skeletal muscles at various sites were examined with special reference to the fluctuation of intracellular AChE activity. AChE-rich granules in each muscle throughout the whole body of chick embryos were observed. Since the distribution pattern of these granules changed regularly in the course of the muscle fiber development, advances of muscle differentiation in various sites of the body were compared. (1) The process of muscle development is more advanced in the trunk muscles than in the limb muscles. (2) The dorsal trunk muscles differentiate one day earlier than the ventral ones. (3) Within the same limb, proximal muscles differentiate approximately 24 hr ahead of distal ones. (4) The development of posterior limb muscles advances faster than that of anterior limb muscles. (5) Within the thigh muscles, the flexor muscles tend to differentiate earlier than the extensor muscles.     

The fatty acid-binding protein from human skeletal muscle

Fatty acid-binding protein (FABP) was isolated from human skeletal muscle by gel filtration and anion- and cation-exchange chromatography. The isolation procedure, however, with rat and pig skeletal muscle gave mostly inactive preparations. Rat muscle FABP preparations contained parvalbumin as a contaminant. FABP from human muscle had a Mr of about 15 kDa, a pI value of 5.2, and a Kd value with oleic acid of 0.50 microM. Skeletal muscle and heart FABPs and their antisera showed a strong cross-reactivity on Western blots and in enzyme-linked immunosorbent assays (ELISA). No cross-reactivity was observed with liver FABP and its antiserum. On the basis of amino acid composition, electrophoretic behavior, fatty acid binding, and immunochemical properties, human skeletal muscle FABP must be similar or closely related to human heart FABP. The FABP content determined by ELISA was comparable in various human muscles and cultured muscle cells, but lower than that in rat muscles.     

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.     

Localization of surface and internal acetylcholine receptors in developing fast and slow muscles of the chick embryo

The localization of surface and internal acetylcholine (ACh) receptors was investigated in the developing anterior and posterior latissimus dorsi (ALD and PLD) muscles in the chick embryo (11, 15, and 19 days) by autoradiography using ¹²⁵I-α-bungarotoxin (BTX). At 11 days, ACh receptors were already preferentially at neuromuscular junctions. Internal ACh receptors, measured using muscles made permeable to BTX by saponin treatment, were scattered throughout the length of each muscle fiber with or without a slight increase in their number around neuromuscular junctions. Quantitative analysis of grains in montage electron micrographs of muscle fibers from 11-day embryos revealed that intracellular specific BTX binding sites were the Golgi complex and multivesicular bodies. The number of silver grains over the Golgi complex decreased greatly after puromycin treatment of organ-cultured muscles. These findings strongly suggest that the Golgi complex is one of the sites involved in the production of ACh receptors in the skeletal muscle cells in vivo. Multivesicular bodies are assumed to be involved in the degradation of ACh receptors.     

Myoneural junctions and neurosecretory endings on spermathecal muscle fibers of two weevils,Sitophilus granarius and Hypera postica

The fine structure of muscle fibers connecting the two arms of the spermatheca and their innervating axons was studied with the electron microscope. The muscle fibers appear to be a sub-set of skeletal and not visceral muscles. Neurosecretory axons with electron dense granules are adjacent to the muscle fibers in young females O-day post-eclosion but not in the ovipositing adult. The typical nerves form synaptic junctions with muscle fibers at all ages but the nerves are divided into two types based on the morphology of the synaptic vesicles they contain, either spherical or flattened.     

T system in ascidian muscle: organization of the sarcotubular system in the caudal muscle cells of Botryllus schlosseri tadpole larvae.

The organization of the sarcotubular system has been examined in the caudal muscle cells of the ascidian. Botryllus schlosseri. At variance with striated muscle of other protochordates. Botryllus muscle cells are endowed with a well-developed T system, which has a peculiar laminar structure. The thin T laminae are in continuity with the plasma membrane and extend longitudinally in the intermyofibrillar spaces. At the level of the I-band the T laminae are focally associated with SR cisternae in dyad junctions similar to those observed in invertebrate muscles. These findings are discussed in relation to the origin of the sarcotubular system in vertebrate muscle.     

Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.     

Polymorphic forms of troponin T and troponin C and their localization in striated muscle cell types

1. 1. Immunochemical studies have shown that the major forms of troponin T present in fast skeletal, slow skeletal and cardiac muscles are different proteins.

2. 2. Similar studies indicate that the major form of troponin C present in fast skeletal muscles differs from troponin C present in slow skeletal and cardiac muscle cells. The forms of troponin C present in slow skeletal and cardiac muscles are immunochemically very similar.

3. 3. The antibodies to the polymorphic forms of troponin T and troponin C are specific for the muscle type, except in the case of the slow skeletal and cardiac muscle forms of troponin C.

4. 4. By the immunoperoxidase technique, it has been shown that the fast skeletal muscle troponin T is localized in type II cells and slow skeletal muscle troponin T in type I cells.

5. 5. Fast skeletal muscle troponin C is present in type II cells and a different troponin C, identified by its reaction with the antibody against cardiac troponin C, is present in type I cells.

6. 6. It is concluded that in normal adult skeletal muscle, fast muscle forms of troponin I, troponin T and troponin C are present together as a homocomplex in type II cells and the slow muscle forms exist as an analagous homocomplex in type I cells.