Supramolecular organization of the subsarcolemmal cytoskeleton of adult skeletal muscle fibers. A review

This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.     

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.     

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.     

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.     

Association of cytoskeletal proteins with newly formed acetylcholine receptor aggregates induced by embryonic brain extract

Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.     

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.     

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.     

Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [35S]methionine or [35S]sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 125I-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord. During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.     

Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo

The synaptic basal lamina, a component of extracellular matrix (ECM) in the synaptic cleft at the neuromuscular junction, directs the formation of new postsynaptic specializations, including the aggregation of acetylcholine receptors (AChRs), during muscle regeneration in adult animals. Although the molecular basis of this phenomenon is unknown, it is mimicked by AChR-aggregating proteins in ECM-enriched fractions from muscle and the synapse-rich electric organ of the ray Torpedo californica. Molecules immunologically similar to these proteins are concentrated in the synaptic basal lamina at neuromuscular junctions of the ray and frog. Here we demonstrate that immunologically, chemically, and functionally similar AChR-aggregating proteins are also associated with the ECM of several other tissues in Torpedo. Monoclonal antibodies against the AChR-aggregating proteins from electric organ intensely stained neuromuscular junctions and the ventral surfaces of electrocytes, structures with a high density of AChRs. However, they also labeled many other structures which have basal laminae, including the extrajunctional perimeters of skeletal muscle fibers, smooth and cardiac muscle cells, Schwann cell sheaths in peripheral nerves, walls of some blood vessels, and epithelial basement membranes in the gut, skin, and heart. Some structures with basal laminae did not stain with the antibodies; e.g., the dorsal surfaces of electrocytes. Bands of similar molecular weight were detected by the antibodies on Western blots of extracts of ECM-enriched fractions from electric organ and several other tissues. Proteins from all tissues examined, enriched from these extracts by affinity chromatography with the monoclonal antibodies, aggregated AChRs on cultured myotubes. Thus, similar AChR- aggregating proteins are associated with the extracellular matrix of many Torpedo tissues. The broad distribution of these proteins suggests they have functions in addition to AChR aggregation.     

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.     

Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina

Basal lamina (BL) ensheathes each skeletal muscle fiber and passes through the synaptic cleft at the neuromuscular junction. Synaptic portions of the BL are known to play important roles in the formation, function, and maintenance of the neuromuscular junction. Here we demonstrate molecular differences between synaptic and extrasynaptic BL. We obtained antisera to immunogens that might be derived from or share determinants with muscle fiber BL, and used immunohistochemical techniques to study the binding of antibodies to rat skeletal muscle. Four antisera contained antibodies that distinguished synaptic from extrasynaptic portions of the muscle fiber's surface. They were anti- anterior lens capsule, anti-acetylcholinesterase, anti-lens capsule collagen, and anti-muscle basement membrane collagen; the last two sera were selective only after antibodies binding to extrasynaptic areas had been removed by adsorption with connective tissue from endplate-free regions of muscle. Synaptic antigens revealed by each of the four sera were present on the external cell surface and persisted after removal of nerve terminal. Schwann cell, and postsynaptic plasma membrane. Thus, the antigens are contained in or connected to BL of the synaptic cleft. Details of staining patterns, differential susceptibility of antigens to proteolysis, and adsorption experiments showed that the antibodies define at least three different determinants that are present in synaptic but not extrasynaptic BL.     

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.     

Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction.

The nicotinic acetylcholine receptor (AChR) from the electric organ of T. californica is highly phosphorylated on tyrosine residues in vivo. In contrast, tyrosine phosphorylation of the AChR in rat myotube cultures is barely detectable. To determine whether this low level of tyrosine phosphorylation of the AChR in muscle cell cultures is due to a lack of neuronal innervation, we examined tyrosine phosphorylation of the AChR in rat diaphragm in vivo. Immunofluorescent double labeling of cryostat sections of rat diaphragm using antibodies specific for phosphotyrosine or the AChR showed a direct colocalization of phosphotyrosine with the AChR at the neuromuscular junction. Using anti-phosphotyrosine antibodies, immunoblots of AChR partially purified from rat diaphragm demonstrated that the rat AChR contains high levels of phosphotyrosine. Denervation of rat diaphragm induced a time-dependent decrease in tyrosine phosphorylation of the AChR, as measured by immunocytochemical and immunoblot techniques. Tyrosine phosphorylation of the AChR occurred late in the development of the neuromuscular junction, between postnatal days 7 and 14. These studies suggest that muscle innervation regulates tyrosine phosphorylation of the AChR and that tyrosine phosphorylation may play an important role in the developmental regulation of the AChR.     

Co-localization of nitric oxide synthase I (NOS I) and NMDA receptor subunit 1 (NMDAR-1) at the neuromuscular junction in rat and mouse skeletal muscle

Nitric oxide synthase I (NOS I) has been localized to the skeletal muscle sarcolemma in a variety of vertebrate species including man. It is particularly enriched at neuromuscular junctions. Recently, the N-methyl-d-aspartate (NMDA) receptor subunit 1 (NMDAR-1) has been detected in the postjunctional sarcolemma of rat diaphragm, providing a clue as to the possible source of Ca²⁺ ions that are necessary for NOS I activation. To address this possibility, we studied the distribution of NMDAR-1 and NOS I in mouse and rat skeletal muscles by immunohistochemistry and enzyme histochemistry. NMDAR-1 and NOS I were closely associated at neuromuscular junctions primarily of type II muscle fibers. NOS I was also present in the extrajunctional sarcolemma of this fiber type. Dystrophin, β-dystroglycan, α-sarcoglycan, and spectrin were found normally expressed in both the junctional and extrajunctional sarcolemma of both fiber types. By contrast, in the muscle sarcolemma of MDX mice, dystrophin and dystrophin-associated proteins were reduced or absent. NOS I immunoreactivity was lost from the extrajunctional sarcolemma and barely detectable in the junctional sarcolemma. NOS I activity was clearly demonstrable in the junctional sarcolemma by NADPH diaphorase histochemistry, especially when the two-step method was used. NMDAR-1 was not altered. These data suggest that different mechanisms act to attach NOS I to the junctional versus extrajunctional sarcolemma. It may further be postulated that NMDA receptors are involved not only in the regulation but also sarcolemmal targeting of NOS I at neuromuscular junctions of type II fibers. The evidence that glutamate may function as a messenger molecule at vertebrate neuromuscular junction is discussed.     

Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle

In adult vertebrate skeletal muscle acetylcholine receptors are localized to the neuromuscular junction. Upon denervation, this distribution changes, with new receptors appearing in extrajunctional regions of the muscle fiber. The location of acetylcholine receptors in innervated or denervated muscle may result, in part, from the distribution of their RNAs. This was tested by assaying for receptor RNAs in junctional and extrajunctional regions of innervated and denervated rat soleus muscle using in situ hybridization and RNAase protection assays. These experiments showed alpha, beta, and delta subunit RNAs concentrated beneath the endplates of innervated muscle fibers. Following denervation, there was an unequal distribution of receptor RNAs along the muscle fiber, with highest levels occurring in extrajunctional regions near the endplate. These data are consistent with a nonuniform pattern of gene expression in adult skeletal muscle fibers.     

Talin is a post-synaptic component of the rat neuromuscular junction

Talin is a protein, recently discovered in chicken gizzard, which occurs at sites of actin-plasma membrane interaction in several cell types. Vinculin also occurs at many of these sites, possibly in association with talin. In this study, three antisera against talin were used to probe the neuromuscular junction of rat skeletal muscle, which is also a site of vinculin accumulation. By immunofluorescence, all three sera stained the junction strongly in frozen sections of rat diaphragm. The extrajunctional periphery was lightly and irregularly stained in some muscle cells; others seemed not to be stained outside the junction. Staining remained at junctions and increased in extrajunctional regions of muscle denervated 6 weeks before sacrifice. The staining in all cases was abolished by competition with purified talin. One serum tested by immunoblotting recognized one protein at Mr 215 000 (identical with the value for chicken gizzard talin) and traces of a second at Mr 190 000 (corresponding to a known proteolytic fragment of talin). We conclude that rat muscle talin is similar in its general protein structure to chicken gizzard talin, and is a post-synaptic component of the neuromuscular junction.     

Role of Activity in Determining Properties of the Neuromuscular System in Crustaceans

SYNOPSIS. Crustacean muscle fibers, like those of higher vertebrates,are diversified in physiology, morphology, and biochemical attributes.However, unlike motor units of mammals, those of crustaceansusually do not contain fibers of uniform type. Motor neuronactivity acts as a unifying force for the motor units of mammalianmuscles, but its role in determining properties of crustaceanmotor units is less well defined. In certain crustacean muscles,differential activity of sensory-motor systems is importantfor establishing muscle fiber properties during early development.In freshwater crayfish, neuromuscular junctions of a phasicmotor neuron are altered physiologically and morphologicallyby chronic stimulation; the adapted junctions release less transmitterper impulse and are more fatigue-resistant than naive junctions.The muscle fibers may also adapt to chronic stimulation, butless dramatically and at a slower rate. The adaptive responsesof the neuromuscular junction can be achieved through manipulationof sensory input and with little increase in motor impulse activity.This suggests that altered protein synthesis is triggered centrallyby synaptic input to the motor neuron. In general, present evidencesuggests that long-term adaptation of neuromuscular junctionsand muscle fibers of crustaceans can occur in response to alteredactivity in the nervous system, in spite of the fact that certainmuscle fiber properties appear to be genetically predetermined.Some aspects of matching between neuromuscular junction andmuscle fiber appear to be determined in response to growth ofthe muscle fiber; other features are activity-dependent; andsome may result from expression of inherent neuronal properties.     

Cytoskeletal protein contents before and after hindlimb suspension in a fast and slow rat skeletal muscle

Transversal cytoskeletal organization of muscle fibers is well described, although very few data are available concerning protein content. Measurements of desmin, alpha-actinin, and actin contents in soleus and extensor digitorum longus (EDL) rat skeletal muscles, taken with the results previously reported for several dystrophin-glycoprotein complex (DGC) components, indicate that the contents of most cytoskeletal proteins are higher in slow-type fibers than in fast ones. The effects of hypokinesia and unloading on the cytoskeleton were also investigated, using hindlimb suspension. First, this resulted in a decrease in contractile protein contents, only after 6 wk, in the soleus. Dystrophin and associated proteins were shown to be reduced for soleus at 3 wk, whereas only the dystrophin-associated proteins were found to increase after 6 wk. On the other hand, the contents of DGC components were increased for EDL for the two durations. Desmin and alpha-actinin levels were unchanged in the same conditions. Consequently, it can be concluded that the cytoskeletal protein expression levels could largely contribute to muscle fiber adaptation induced by modified functional demands.