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.     

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.     

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross- linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.     

A Role of Tyrosine Phosphatase in Acetylcholine Receptor Cluster Dispersal and Formation

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor–coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot–associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.     

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.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Isolated primary osteocytes express functional gap junctions in vitro

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed “prepatterning”. The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These “active” contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.     

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.     

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.     

The role of lateral migration in the formation of acetylcholine receptor clusters induced by basic polypeptide-coated latex beads

During the formation of the neuromuscular junction, the nerve induces the clustering of acetylcholine receptors (AChR) in the postsynaptic membrane. This process can be mimicked by treating cultured Xenopus myotomal muscle cells with basic polypeptide-coated latex beads. Using this bead-muscle coculture system, we examined the role of lateral migration of AChRs in the formation of the clusters. First, we studied the contributions of the preexisting and newly inserted AChRs. After the cluster formation was triggered by the addition of the beads, preexisting receptors were immediately recruited to the bead-muscle contacts and they remained to be the dominant contributor during the first 24 hr. New AChRs, which were inserted after the addition of the beads, appeared at the clusters after a 4-hr delay and, thereafter, there was a steady increase in their contribution. After 24-48 hr, newly inserted AChRs could be detected at the bead-induced clusters to the same extent as the preexisting AChRs. During this period, new receptors were continuously inserted into the plasma membrane, but there was no evidence of a local insertion at sites of new cluster formation. Concanavalin A (Con A) at a concentration of 100 micrograms/ml caused a fivefold decrease in the fraction of mobile AChRs and a large decrease in their diffusion coefficient. Pretreatment of cells with Con A suppressed clustering of preexisting AChRs, but left intact the contribution of the mobile newly inserted AChRs. Succinyl Con A, the divalent derivative of Con A which affected the mobility to a much less extent than Con A, had little effect on the clustering process. These results show that the formation of AChR clusters in Xenopus is mediated by lateral migration of AChRs within the plasma membrane and are consistent with the diffusion-trap hypothesis, which depicts freely diffusing AChR aggregating at the bead-muscle contacts where they bind to other localized molecular specializations induced by the beads.     

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.     

Development of acetylcholinesterase induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells

The formation of acetylcholine receptor (AChR) clusters can be induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells. Here we investigated the development of acetylcholinesterase (AChE) at the bead-induced AChR clusters. AChE activity began to appear at the clusters after 1 day of bead-muscle coculture and was present at all of the bead-induced clusters within 4-7 days. Electron microscopy revealed that AChE reaction products were discretely localized within the cleft and the membrane invaginations at the bead-muscle contacts. Thus, the beads can mimic the nerve in inducing a local accumulation of both the AChRs and AChE, suggesting that the development of both specializations can be effected by a common stimulus.     

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture

Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.     

Effects of innervation on the distribution of acetylcholine receptors in regenerating skeletal muscles of adult chickens

In order to determine the roles of nerves in the formation of clusters of acetylcholine receptors (AChRs) during synaptogenesis, we examined the distribution of AChRs in denervated, nerve-transplanted (neurotized) muscles and in regenerated skeletal muscles of adult chickens by fluorescence microscopy using curaremimetic toxins. In the denervated muscles, many extrajunctional clusters developed at the periphery of some of the muscle nuclei of a single muscle fiber and continued to be present for up to 3 months. The AChR accumulations originally present at the neuromuscular junctions disappeared within 3 weeks. In the neurotized muscles, line-shaped AChR clusters developed at 4 days after transection of the original nerve, but no change in the distribution of AChRs had occurred even at 2 months after implantation of the foreign nerve. The line-shaped AChR clusters were found to be newly formed junctional clusters as they were associated with nerve terminals of similar shape and size. Some of both the line-shaped and extrajunctional clusters were formed at least partly by the redistribution of preexisting AChRs. Finally, based on the above observations, the regenerating muscle fibers in normal muscles and in denervated muscles were examined: The extrajunctional clusters appeared in both kinds of muscles at 2 weeks after injury. Afterward, during the innervation process, the line-shaped AChR clusters developed while the extrajunctional clusters disappeared in the innervated muscles. In contrast with this, in the absence of innervation, only the extrajunctional clusters continued to be present for up to 3 months. These results demonstrate clearly that the nerve not only induces the formation of junctional clusters at the contact site, but also prevents the formation of clusters at the extrajunctional region during synaptogenesis.     

Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions

When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.     

A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells

During the development of the neuromuscular junction, acetylcholine receptors (AChRs) become clustered in the postsynaptic membrane in response to innervation. In vitro, several non-neuronal stimuli can also induce the formation of AChR clusters. DC electric field (E field) is one of them. When cultured Xenopus muscle cells are exposed to an E field of 5-10 V/cm, AChRs become clustered along the cathode-facing edge of the cells within 2 h. Recent studies have suggested the involvement of tyrosine kinase activation in the action of several AChR clustering stimuli, including nerve, polymer beads, and agrin. We thus examined the role of tyrosine phosphorylation in E field-induced AChR clustering. An antibody against phosphotyrosine (PY) was used to examine the localization of PY-containing proteins in E field-treated muscle cells. We found that anti-PY staining was colocalized with AChR clusters along the cathodal edge of the cells. In fact, cathodal PY staining could be detected before the first appearance of AChR clusters. When cultures were subjected to E fields in the presence of a tyrosine kinase inhibitor, tyrphostin RG-50864, cathodal AChR clustering was abolished with a half maximal inhibitory dosage of 50 microM. An inactive form of tyrphostin (RG-50862) had no effect on the field-induced clustering. These data suggest that the activation of tyrosine kinases is an essential step in E field-induced AChR clustering. Thus, the actions of several disparate stimuli for AChR clustering seem to converge to a common signal transduction mechanism based on tyrosine phosphorylation at the molecular level.     

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.     

Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.     

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.     

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.