Electron cytochemical study of the muscle cell surface

Summary The lectins wheat germ agglutinin and limulus polyphemus were used as cytochemical probes to study the ultrastructural localization of sialic acid at the cell surface of rat muscle fibers. In addition cytochemical studies employing strontium as an electron-dense marker were also carried out to investigate cation binding sites at the muscle cell surface. The results showed binding of the lectins to the glycocalyx, caveolae and the basal lamina of the muscle fibers. These binding sites matched the ones observed in the cytochemical studies using strontium as a marker. Based on these observations we suggest that the glycocalyx, caveolae and the basal lamina of the muscle fiber may be involved in the binding of Ca⁺⁺ and that significant amounts of Ca⁺⁺ may be normally present at the muscle cell surface.Supported by a grant from the Muscular Dystrophy Association and by Center Grant NS-1176 from the National Institute of Neurological and Communicative Disorders and Stroke     

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.     

Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.     

Changes in the content and distribution of sialic acid on the basal surface of isoproterenol-stimulated mouse parotid acinar cells

The presence, distribution and content of sialic acid on the cell surface in collagenase-dispersed acini obtained both from unstimulated as well as from in vivo isoproterenol-stimulated mouse parotid have been studied. To this end, sialic acid residues have been qualitatively and quantitatively analyzed by 1) cytochemical labeling by wheat germ agglutinin (WGA), 2) biochemical procedures and 3) isotopic labeling by [3H]WGA (WGA-N-[acetyl-3H]-acetylated). Electron microscopy revealed striking differences in the binding of ferritin-conjugated WGA at the basal, lateral and apical cell surface. Unstimulated acinar cells showed a heavy patch-distributed binding of ferritin-conjugate on the basal cell surface while it was homogeneous and very scarce on the lateral one and absent on the apical cell surface. During the first few hours after isoproterenol, the WGA binding sites at the basal cell surface became homogeneously distributed. This fact was coincident with a loss of about 60 to 70% both in the content of neuraminidase-releasable sialic acid and in the binding of [3H]WGA to the acinar surface. These findings suggest that the release of sialic acid as free residues, which has been involved in the isoproterenol-triggered cell proliferation-inducing mechanism in the mouse parotid, would occur at the glycocalyx corresponding to the basal plasma membrane of the acinar cells.     

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold- particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin- binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.     

Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve

We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.     

Acetylcholinesterase clustering at the neuromuscular junction involves perlecan and dystroglycan.

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve- muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.     

Subcellular distribution of ionic components in gastric mucosa of the guinea pig

Summary The topographical distribution of cations, anions and polyanions in the guinea-pig stomach has been studied by ultrastructural cytochemical methods. After fixation with the pyroantimonate-osmium tetroxide solution, variable-sized precipitates were localized in the basolateral extracellular space bordering parietal cells or chief cells but not in that bordering mucus-secreting cells. The basal lamina of all gastric cells disclosed a continuous layer of heavy antimonate deposits. Parietal cells disclosed uniformly fine deposits also on the apical plasmalemma both at the main lumen and in the intracellular canaliculi, and revealed, as well, coarse precipitates in the mitochondria. Fixation with a silver acetate-osmium tetroxide solution yielded nitric acid-resistant, silver deposits confined to the luminal surface of the apical plasmalemma in the main lumen and intracellular canaliculi, the lateral intercellular space, the outer surface of the basal plasmalemma and the basal lamina of the parietal cell.Staining with dialyzed iron demonstrated a glycocalyx rich in acid mucosubstance on the basolateral plasmalemma but not on the apical plasmalemma of parietal cells. In contrast, acid glycoconjugate was visualized on the apical plasmalemma of isthmus cells, mucous neck cells and the transitional cell between isthmus and mucous neck cells but little or no acidic glycoconjugate was demonstrated on the basolateral plasmalemma of these cells. The entire plasmalemma of gastroendocrine cells, unlike other epithelial cells, stained uniformly for acidic glycoconjugate. The dialyzed iron and high iron diamine methods stained the outer compartment of mitochondria in parietal cells intensely and that in other gastric cells lightly. These reagents stained the basal lamina of all gastric cells as did ruthenium red. The several characteristic cytochemical properties of parietal cells presumably relate to the unique secretory activity of these cells and are consistent with the view of the intracellular canaliculi of the parietal cell as the main route for hydrogen and chloride ion secretion.     

Receptors to agglutinin fromDolichus biflorus (DBA) at the synaptic basal lamina of rat neuromuscular junction

Summary The binding of agglutinin fromDolichus biflorus (DBA) and other lectins (Concanavalin A, agglutinin from wheat germ and lectin fromBandeiraea simplicifolid) to synaptic and extrasynaptic portions of the basal lamina of muscle fibers, was studied with histochemical methods. In rat muscle, DBA-binding is specifically detected at the basal lamina of neuromuscular junction. However, long-term (6 months) denervated end-plate in adult rat muscle failed to bind DBA. During normal development, synaptic DBA receptors appear later than acetylcholine receptors or acetylcholinesterase at the rat neuromuscular junction. Generalized DBA-binding to motor end-plates is first visualized in 3-day-old rats, but section of sciatic nerve in 1-day-old rats prevents the appearence of synaptic DBA-binding on the leg end-plates. It is suggested, therefore, that the synaptic DBA receptors could be related to the postnatal stabilization of rat neuromuscular synapses.     

Fusion between myogenic cells in Vivo: An ultrastructural study in regenerating murine skeletal muscle

Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.     

High resolution scanning electron microscopy of frog sartorius muscle

A field emission-type scanning electron microscope (SEM) was used to study the three-dimensional ultrastructure of frog sartorius muscles. Various preparative procedures were tested to seeks better specimen preparation for high resolution SEM observation. Procedures should be chosen depending on the information desired. The cell surface and intracellular organization of muscle fibers were best visualized when the tissues were fixed with tannic acid-OsO4 and torn after critical point drying. The basal lamina appeared as a continuous felt-like layer, onto which fine filamentous materials adhered. The true outer surface of the sarcolemma was not seen, whereas the true inner surface was occasionally exposed and exhibited numerous caveolae, membraneous fragments and fine filaments attached to its surface. In freeze-fractured and dried tissues, the cleaved sarcolemma showed numerous apertures of caveolae and T-system tubules. Inside the cell, the myofibrils showed a typical branding pattern of the sarcomere. Thick myofilaments were regularly beaded except for the pseudo-H-zone. Around the myofibrils the sarcoplasmic reticulum and T-system were also clearly observed. The results are discussed with special reference to the usefulness and limitation of the high resolution SEM in studying the ultrastructure of cells and tissues.     

Morphology and histochemistry of the myotendineal junction of the rat calf muscles. Histochemical, immunohistochemical and electron-microscopic study.

The macromolecular composition and morphometry of the myotendineal junction (MTJ) of slow-twitch (type 1) and fast-twitch (type 2) muscle fibers were studied in gastrocnemius-soleus-Achilles unit of the rat. Proteoglycans and glycosaminoglycans, type III collagen, fibronectin and laminin could be detected at the myotendineal junction. Due to the membrane folding finger-like processes were seen at the MTJ. The processes of type 1 fibers were greater in size. However, due to the subdivisions the processes of type 2 muscle fibers had a significantly greater surface length per muscle cell diameter than type 1 fibers. The myotendineal endings of both fiber types had a characteristic basal lamina, which was about three times thicker than in the longitudinal site of the same muscle cells. The basal lamina of type 1 fibers at the MTJ was significantly thicker than that of type 2 fibers.     

Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca(2+) handling

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca(2+)-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca(2+). We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca(2+) channels and Ca(2+) binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca(2+) pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca(2+) pump, or the type 1 inositol 1,4, 5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca(2+) pump to the cell periphery, whereas the SR Ca(2+) pump was located deeper in the cell. The presence of L-type Ca(2+) channels, the PM Ca(2+) pump, and the Ca(2+) bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca(2+) handling.     

Localization of calcium binding sites associated with the calcium spike in barnacle muscle

Summary La ion behaves as a competitive inhibitor of Ca ions on the calcium spike in the giant muscle fiber of the barnacle,Balanus nubilus. La-treated muscle fibers, in which the rate of rise of the spike was diminished to a known degree, have been examined with the electron-microscope. In such fibers dense particles are seen in association with the surface membrane and external lamina of the cell. La particles are not seen in association with fibers that have been allowed to recover from La inhibition before fixation. The number of La particles seen in association with the muscle fiber increases with increasing La concentration when the Ca and Mg concentrations are held constant and decreases with increasing Ca and Mg concentration when the La concentration is held constant. The results suggest that the La visible in the electron-microscope under the conditions of these experiments is bound to a class of sites similar to those involved in the Ca spike.     

Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction

Summary A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.     

Nerve-induced remodeling of muscle basal lamina during synaptogenesis

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.     

Ca++-ATPase in the central nervous system: an EM cytochemical study

Ca++-ATPase plays an important role in regulation of the intracellular Ca++ concentration. Biochemical studies of brain have demonstrated that Ca++-ATPase co-purifies with synaptosomes, with synaptic plasma membrane and synaptic vesicle fractions. To better understand the role of this enzyme in normal brain function, we used an electron microscopic (EM) cytochemical method to determine the localization of Ca++-ATPase in rat brain. Reaction product occurred along cytoplasmic membranes. Specific areas of increased reaction product were seen at many but not all post-synaptic densities. Intracellular Ca++-ATPase reaction product was associated with all synaptic vesicles examined and with the Golgi and smooth endoplasmic reticulum (SER). Unlike the situation in peripheral nerve, Ca++-ATPase at the node of Ranvier in the CNS localized preferentially to the nodal axolemma. The localization of Ca++-ATPase at synaptic vesicles agrees with the biochemical evidence for its localization and with the cytochemical evidence for Ca++-ATPase sequestration in those vesicles. The restricted localization at postsynaptic densities suggests that it may be involved in extrusion of Ca++ at synapses where neurotransmitter release causes Ca++ influx.     

The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.     

Affiliation of large numbers of fibroblasts with larval muscle fibers

Muscle fibers from fourth and fifth instar caterpillars were examined with scanning and thin section electron microscopy. Scanning micrographs showed that early fifth instar specimens had a population of cells lying beneath the basal lamina over the surface of the muscle fiber and in conjunction with tracheoles and nerves. At least two cell types were present. One type could be categorized as tracheoblasts of their close association with the tracheoles and the presence of taenidia within the tracheoblast cytoplasm in sectioned material. A second cell type, characterized by long filamentous processes, contained extensive rough endoplasmic reticulum and cisternae swollen with an electron-dense substance similar in appearance to the basal lamina. This ultrastructural appearance is characteristic of vertebrate fibroblasts and certain types of insect hemocytes. Early and late fourth instar specimens had few cells on their muscle fiber surfaces. Measurements of the basal lamina thickness were taken from thin sections of nondigested muscle fibers of early fourth, late fourth, and early fifth instar animals. The results showed that the basal lamina underwent a large increase in thickness between the fourth and fifth instars. The proliferation of cells which appeared to be in an actively synthesizing state paralleled the increase in basal lamina thickness. This suggests the hypothesis that these cells are active in connective tissue formation, and contribute to the formation of the basal lamina that lies over both them and the muscle fiber.     

Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.