Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed.     

Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures

The effect of denervation on acetylcholine receptor (AChR) cluster distribution on cultured Xenopus muscle cells has been examined in order to study the role of intact nerve in the maintenance of clusters at the nerve-muscle junction during development. AChRs on the muscle cell were labeled with tetramethyl rhodamine-conjugated alpha-bungarotoxin and sequential changes in AChR cluster distribution were examined with a fluorescence microscope using an image intensifier. Denervation was carried out by exposing the nerve cell body to a focused laser light of a high intensity. After this procedure the neurites originating from the cell quickly disintegrated and large AChR clusters associated with nerve divided into smaller clusters. Individual clusters subsequently decreased in size and finally disappeared. In about 30% of the cases new AChR clusters appeared at the extrajunctional region after denervation. These observations indicate that intact nerves are necessary for the maintenance of receptor localization at the nerve-muscle junction and that nerve-induced accumulation is seemingly reversible during the early period of synapse formation. We tested the idea that receptor clusters were lost due to diffusion of receptors in the muscle membrane after denervation. However, the rate of receptor cluster dispersal after denervation was much slower than that predicted by the diffusion model, suggesting that diffusion of receptors is not a rate-limiting step. Furthermore, we found that receptor clusters at the junction stabilize during days in culture. Thus, 80-90% of receptor clusters at the nerve-muscle junction disappeared at 7 hr after denervation in 1-day cocultures, while about 50% of receptor clusters remained after denervation in 3-day cocultures.     

Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes

The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters.     

Neuregulin inhibits acetylcholine receptor aggregation in myotubes

The high local concentration of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction results from their aggregation by the agrin/MuSK signaling pathway and their synthetic up-regulation by the neuregulin/ErbB pathway. Here, we show a novel role for the neuregulin/ErbB pathway, the inhibition of AChR aggregation on the muscle surface. Treatment of C2C12 myotubes with the neuregulin epidermal growth factor domain decreased the number of both spontaneous and agrin-induced AChR clusters, in part by increasing the rate of cluster disassembly. Upon cluster disassembly, AChRs were internalized into caveolae (as identified by caveolin-3). Time-lapse microscopy revealed that individual AChR clusters fragmented into puncta, and application of neuregulin accelerated the rate at which AChR clusters decreased in area without affecting the density of AChRs remaining in individual clusters (as measured by the fluorescence intensity/unit area). We propose that this novel action of neuregulin regulates synaptic competition at the developing neuromuscular junction.     

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.     

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.     

Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve- induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

Regulation of turnover and number of acetylcholine receptors at neuromuscular junctions

The number and metabolic stability of acetylcholine receptors (AChRs) at neuromuscular junctions of rat tibialis anterior (TA) and soleus (SOL) muscles were examined after denervation, paralysis by continuous application of tetrodotoxin to the nerve, or denervation and direct stimulation of the muscle through implanted electrodes. After 18 days of denervation AChR half-life declined from about 10 days to 2.3 days (TA) or 3.6 days (SOL) and after 18 days of nerve conduction block to 3.1 days (TA). In contrast, the total number of AChRs per endplate was unaffected by these treatments. Denervation for 33 days had no further effect on AChR half-life but reduced the total number of AChRs to about 54% (SOL) or 38% (TA) of normal. Direct stimulation of the 33-day denervated SOL from day 18 restored normal AChR stability and counteracted muscle atrophy but had no effect on the decline in AChR number. The results indicate that motoneurons control the stability of junctional AChRs through evoked muscle activity and the number of junctional AChRs through trophic factors.     

Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.     

Synaptic differentiation is defective in mice lacking acetylcholine receptor beta-subunit tyrosine phosphorylation

Agrin activates MuSK, a receptor tyrosine kinase expressed in skeletal muscle, leading to tyrosine phosphorylation of the acetylcholine receptor (AChR) beta-subunit and clustering of AChRs. The importance of AChR beta-subunit tyrosine phosphorylation in clustering AChRs and regulating synaptic differentiation is poorly understood. We generated mice with targeted mutations in the three intracellular tyrosines of the AChR beta-subunit (AChR-beta(3F/3F)). Mice lacking AChR beta-subunit tyrosine phosphorylation thrive postnatally and have no overt behavioral defects, indicating that AChR beta-subunit tyrosine phosphorylation is not essential for the formation of neuromuscular synapses. Nonetheless, the size of synapses and the density of synaptic AChRs are reduced in AChR- beta(3F/3F) mutant mice. Moreover, synapses are structurally simplified and the organization of postjunctional folds is aberrant in mice lacking tyrosine phosphorylation of the AChR beta-subunit. Furthermore, mutant AChRs cluster poorly in response to agrin and are readily extracted from the cell surface of cultured myotubes by non-ionic detergent. These data indicate that tyrosine phosphorylation of the AChR beta-subunit has an important role in organizing AChRs and regulating synaptic differentiation.     

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.     

Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.     

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.     

The localization of acetylcholine receptor clusters in areas of cell-substrate contact in cultures of rat myotubes

We have used interference reflection and fluorescence microscopy to investigate the relationship between cell-substrate contact and the location of clusters of acetylcholine receptors (AChRs) in cultures of rat myotubes. We have found that AChR clusters on the ventral myotube surfaces are always located within broad regions of close cell-substrate contact. Detailed analysis of the fine structure of the AChR cluster and its associated contact region showed that AChRs within a cluster are concentrated between the points of closest cell-substrate apposition. Vinculin, a recently discovered intracellular smooth muscle protein, is also concentrated in broad regions of close contact, interdigitating with AChRs within the clusters.     

Development of rat soleus endplate membrane following denervation at birth

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.     

The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stability

The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth.     

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.     

Sciatin: a myotrophic protein increases the number of acetylcholine receptors and receptor clusters in cultured skeletal muscle

Factors present in neural extracts or in media conditioned by neurons have been shown by others to increase both the number of acetylcholine receptors (AChRs) and the number of receptor clusters in cultures of embryonic skeletal muscle. We have recently shown that the glycoprotein, sciatin, exerts trophic effects on developing muscle in vitro. In the present study, we investigated the effect of sciatin on AChRs in aneural cultures of chick skeletal muscle. Sciatin caused a significant increase in the number of AChRs/dish as measured by binding of ¹²⁵I-α-bungarotoxin (α-Btx) and in acetylcholinesterase (AChE) activity/dish in differentiating muscle cells. The increase in AChRs elicited by sciatin was due solely to increased receptor synthesis and incorporation. The rate of AChR synthesis in sciatin-treated cultures was as much as five times the control rate and was significantly reduced by cycloheximide (10 μM). AChR degradation was unaffected by the myotrophic protein. Although the number of AChRs/dish was increased by sciatin during myogenesis, AChR specific activity, expressed as picomoles ¹²⁵I-α-Btx bound/mg cell protein, was only transiently increased by the myotrophic protein. This contrasted with AChE specific activity in sciatin-treated cultures which remained elevated throughout differentiation. Autoradiographs of ¹²⁵I-α-Btx-labeled cultures showed that sciatin caused an increase in the number and size of AChR “hot spots” and maintained the integrity of these AChR clusters in aneural muscle cultures for up to 5 weeks. At this time control cultures had completely degenerated. The mechanism by which sciatin enhanced the synthesis of AChRs appeared to be distinct from that of tetrodotoxin (TTX), an agent which abolishes muscle activity. However, like theophylline, sciatin might evoke increased synthesis of AChRs via regulation of cyclic AMP since the myotrophic protein increased cAMP both in cells and in conditioned medium. The results of this study suggest that sciatin may be related to the diffusible factor(s) from motor neurons described by others which has trophic effects on AChRs. Furthermore, we suggest that this myotrophic protein may be responsible for the clustering of AChRs and maintenance of receptor clusters at neuromuscular junctions in developing avian muscle.     

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve–muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic cluster of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline–treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline–inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve–muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve-and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve–muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted. © 1995 John Wiley & Sons, Inc.