Increased endocytosis of acetylcholine receptors by dystrophic mouse myotubes in vitro

Multinucleated myotubes, grown in vitro from satellite cells of dystrophic mice (C57BL/6J/dydy) exhibit a reduced sensitivity to ACh. This reduction correlates with a reduced density of 125I-alpha-bungarotoxin (125I-BTX) binding sites on the surface of dystrophic myotubes. Denervated adult muscle fibers from dystrophic mice respond to Ach similarly to denervated normal muscle fibers. Furthermore, cultured dystrophic myotubes, treated with a brain extract which induces AChR clusterization, still show an impaired response to ACh and reduced 125I-BTX binding. Thus AChR function appears altered in dystrophic muscle cells in culture while it appears normal in dystrophic adult muscle, regardless of whether the receptors are dispersed on the membrane or clustered at the junctional site. Metabolic studies on the reduced AChR level in dystrophic myotubes revealed a dramatically reduced half-life (2 vs 10 hr) while the rate of synthesis was unchanged. An increased rate of internalization of AChR was observed in dystrophic myotubes with a corresponding relative increase of the "hidden AChR pool," which could be partially reduced by agents which disrupt the cytoskeleton. No structural alterations could be detected on the AChR molecule as its sedimentation coefficient and subunit composition appeared identical between normal and dystrophic myotubes. Thus the increased turnover of AChR in dystrophic myotubes either reflects subtle alterations of the molecule or a more generalized increase of endocytosis in this form of myopathy.     

Laminin-induced Acetylcholine Receptor Clustering: An Alternative Pathway

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1–induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK−/− mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.     

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin- induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.     

Clathrin-coated membrane: a distinct membrane domain in acetylcholine receptor clusters of rat myotubes

We have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR-rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR-rich domains are independently stabilized.     

Overexpression of rapsyn inhibits agrin-induced acetylcholine receptor clustering in muscle cells

Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of rapsyn cDNA can restore AChR clustering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of rapsyn. A deletion mutant lacking residues 16–254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43^D405,D406) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.     

Formation of complex AChR aggregates in vitro requires α‐dystrobrevin

Efficient function at the neuromuscular junction requires high‐density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. α‐Dystrobrevin (αDB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of αDB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel‐like AChR aggregates on primary muscle cell cultures derived from αDB knockout (αDB‐KO) mice in the absence of nerve or agrin. In myotubes lacking αDB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of αDB are expressed in skeletal muscle: αDB1, αDB1(?), αDB2, αDB2(?), and αDB3. Expression of αDB1 or αDB1(?) in αDB‐KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of αDB2, αDB2(?), αDB3, or an αDB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that αDB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that αDB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in αDB‐KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Mercury decreases the frequency of induced but not spontaneous clustering of acetylcholine receptors

Studies with environmental levels of various metals typically focus on observable neurological symptoms in newborns and adults. Use of the C2C12 skeletal muscle cell line as a developmental model enabled us to test whether environmental insults prevented myotube formation or the assembly of the postsynaptic component of the neuromuscular synapse. Specifically, we asked whether the inorganic metal mercury interfered with the fusion of myoblasts into myotubes, acetylcholine receptor (AChR) clustering, or the agrin signaling events that precede AChR clustering. C2C12 myotubes grown in culture medium containing 10 M mercuric chloride were morphologically indistinguishable from control myotubes at the light-microscopic level, and myoblasts fused into myotubes normally. However, myotubes pretreated with mercury demonstrated a decreased frequency of AChR clustering induced by agrin and other experimental manipulations. Furthermore, mercury pretreatment decreased the agrin-induced tyrosine phosphorylation of the AChR subunit, thus inhibiting the agrin signal transduction pathway. In contrast, mercury failed to decrease the frequency of spontaneous AChR clustering, suggesting that spontaneous AChR clustering differs from agrin-induced AChR clustering in some significant way.This work was supported in part by Midwestern University     

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle. © 1995 John Wiley & Sons, Inc.     

The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains

We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to the substrate. We partially purified AChR clusters by extracting cultured rat myotubes with the cholesterol-specific detergent, saponin. The lipid bilayer of the cluster preparation was analyzed biochemically and the substructure of the bilayers was studied morphologically using the fluorescent probes, dansyl polymyxin B, and 3,3'-di(C12H25 and C18H37) indocarbocyanine iodide (C12- and C18-diI). Our results demonstrate that preparations of AChR clusters have a lipid composition biochemically similar to that of the surrounding plasma membrane. Morphologically, however, the lipid bilayer appears to be arranged into domains that resemble the interdigitating pattern seen for the AChR. This distinctive lipid organization is not due to the use of saponin to purify clusters, as we obtained similar results with clusters isolated by physically shearing myotube cultures. The domain-like organization of the bilayer at clusters is disrupted by treatments that disperse AChR clusters in intact myotubes or that remove peripheral membrane proteins from isolated clusters. This suggests that such proteins may contribute to the organization of the bilayer. Two additional factors may also contribute to the organization of the bilayer: physical constraints imposed by sites of substrate attachment and, to a lesser extent, "boundary" lipid associated with AChR.     

Lipid domains of acetylcholine receptor clusters detected with saponin and filipin

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube- substrate contact (Bloch, R.J., and B. Geiger, 1980, Cell, 21:25-35). We have used two cholesterol-specific cytochemical probes, saponin and filipin, to investigate the lipid nature of these membrane domains. When studied with freeze-fracture electron microscopy or fluorescence microscopy, these reagents reacted moderately and preferentially with the AChR-rich domains of AChR clusters. Little or no reaction with the membrane in "contact" domains was seen. In contrast, membrane regions surrounding the AChR clusters reacted extensively with filipin. These results suggest that, in rat myotubes, the composition or the state of the lipids differs between the two membrane domains of the AChR clusters, and between clusters and surrounding membrane. In chick myotubes, AChR clusters do not appear to react with filipin or saponin, although surrounding membrane reacts extensively with these reagents.     

The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane

The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We show that myotubes differentiated from dystroglycan-/- embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not -/- myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.     

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti- phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.     

Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism

The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non-innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.     

Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2

At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.     

Effects of antibodies to neural cell adhesion molecule (N-CAM) on the differentiation of neuromuscular contacts between ciliary ganglion neurons and myotubes in vitro

Previous experiments have suggested that the neural cell adhesion molecule (N-CAM) may have a role in initial nerve-muscle adhesion. To determine whether N-CAM might be involved in synaptic differentiation, we grew ciliary ganglion neurons and embryonic myotubes together in the presence and absence of monovalent antibodies to N-CAM. In normal cultures, undifferentiated neurites contact myotubes, and the nerve at some of these neurite-myotube contacts acquires concentrations of synaptic vesicle antigens. Most of these vesicle antigen-positive contacts become associated with patches of acetylcholine receptor (AChR) on the surface of the underlying myotube. Contacts without concentrations of vesicle antigens do not become associated with AChR patches. In the presence of antibodies to N-CAM, adhesion between neuronal somata and myotubes was reduced, but neurites contacted myotubes with near-normal frequency. The subsequent differentiation of nerve and muscle at these contacts, as assayed by the localization of vesicle antigens and AChR, proceeded normally in the presence of anti-N-CAM antibodies. The results suggest that N-CAM-mediated adhesion between neurite and myotube is not required for synaptic differentiation.     

Acetylcholine receptor channel properties in rat myotubes exposed to forskolin

Rat myotubes exposed to forskolin were studied by patch-clamp technique in cell-attached single channel recording configuration. Channel open time and opening frequency of the main class of acetylcholine receptor- (AChR-) channels (accounting for more than 90% of all unitary events) decreased in the presence of forskolin (20-100 microM). The forskolin-induced action on the AChR function fully developed with a delay of 30-60 minutes from the peak of cytosolic cyclic AMP (cAMPi) concentration. Both cAMP (1 mM), applied intracellularly for 10 min, and dibutyryl cAMP (0.5 mM), applied extracellularly for 90 min, did not accelerate the rate of desensitization of myotubes studied in whole-cell patch-clamp recording configuration. It was concluded that the action of forskolin on AChR-channel function of rat myotubes could be not associated with the cAMPi-dependent phosphorylation of AChR.     

Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro

During differentiation of embryonic chick skeletal muscle in culture, elaboration of acetylcholine receptor (AChR) and acetylcholinesterase occurs shortly after myoblast fusion. During further development, AChR was found to decrease markedly on the myotube surface, while acetylcholinesterase continued to increase. Surface distribution of AChR, as followed by autoradiography using ¹²⁵I-α-bungarotoxin, was homogeneous in newly fused myotubes. With further differentiation, clusters of AChR appeared on the surface of the myotubes, and their subsequent disappearance paralleled a decrease in overall AChR levels. Quantitative autoradiography showed a reduction of over 75% in the density of AChR on the surface of well differentiated, cross-striated myotubes. Thus the appearance of AChR on the cell surface, its condensation into clusters, and finally its depletion seem to be sequential events in the differentiation of skeletal muscle in culture in the absence of direct neuronal influence.     

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.