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.     

The actin-driven movement and formation of acetylcholine receptor clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.     

Association of cortactin with developing neuromuscular specializations

During the development of the neuromuscular junction (NMJ), motoneurons grow to the muscle cell and the nerve–muscle contact triggers the development of both presynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Previous studies have shown that the activation of tyrosine kinases and the local assembly of an actin-based cytoskeletal specialization are involved in the development of both types of specializations. To understand the link between tyrosine phosphorylation and the assembly of the cytoskeleton, we examined the localization of cortactin in relationship to synaptic development. Cortactin is a 80/85 kD F-actin binding protein and is a substrate for tyrosine kinases. It contains a proline-rich motif and an SH3 domain and is localized at sites of active F-actin assembly. Using a monoclonal antibody against cortactin, its localization at developing NMJs in culture was observed. To understand the spatial and temporal relationship between cortactin and developing synaptic structures, cultured muscle cells and spinal neurons from Xenopus embryos were treated with beads coated with heparin-binding growth-associated molecule to induce the formation of AChR clusters and SV clusters and the localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells were treated with beads, cortactin became localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association was most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization could be detected as early as 10 min after the bead-neurite contact was established. Cortactin-enriched contacts later showed concentration of F-actin (at 1 h) and clusters of SVs (at 24 h). These data suggest that cortactin mediates the local assembly of the cytoskeletal specialization triggered by the synaptogenic signal on both nerve and muscle.     

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.     

Increase in intracellular calcium induced by the polycation-coated latex bead, a stimulus that causes postsynaptic-type differentiation in cultured Xenopus muscle cells

The polycation-coated latex bead is a potent stimulus for the induction of postsynaptic-type differentiation in cultured Xenopus myotomal muscle cells. Specializations characteristic of the neuromuscular junction, such as clusters of acetylcholine receptors and other postsynaptic-specific proteins, develop at the bead-muscle contact. Previous studies have shown that a deprivation of extracellular calcium inhibits the effect of the beads in causing the development of these specializations. This suggests that an increase in intracellular Ca2+ is a necessary condition for the development of this specialization. In this study, we tested whether an increase in intracellular calcium is observable upon the bead-muscle contact. The measurement was carried out on cells loaded with the fluorescent calcium indicator fura-2 AM by digitized video microscopy. When polycation-coated beads were added to cells, an increase in intracellular calcium concentration in the range of 5 to 57% of the resting level was observed within 10 sec after bead-muscle contact. Afterward, the calcium level gradually returned to the resting level with a time course of about 3 min. Uncoated beads, which do not induce the formation of acetylcholine receptor clustering, failed to elicit this calcium transient. Removal of extracellular calcium as well as blocking calcium channels with 50 microM verapamil also suppressed this transient induced by the polycation-coated beads. Both treatments are known to suppress the formation of receptor clusters by these beads. These results suggest that the polycation-coated beads cause an influx of calcium by increasing the membrane conductance to this ion. This process may underlie the signaling of the postsynaptic differentiation.     

Effects of denervation and direct electrical stimulation upon acetylcholine receptors and acetylcholinesterase accumulation in developing latissimus dorsi muscle of the chick

The effects of denervation and of direct electrical stimulation of denervated muscle upon the acetylcholine receptor (AChR) clusters and acetylcholinesterase (AChE) spots in the fast avian muscle posterior latissimus dorsi have been investigated. Denervation at day 2 after hatching leads to a disappearance of the junctional AChR clusters and to a marked decrease of AChE spots. Direct electrical stimulation of denervated muscle allows the maintenance of AChR clusters and partly prevents the loss of AChE spots. When AChR cluster and post-synaptic AChE have disappeared in a denervated muscle, muscle activity induced by direct stimulation is unable to induce their accumulation.     

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.     

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.     

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.     

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.     

Essential roles of the acetylcholine receptor gamma-subunit in neuromuscular synaptic patterning

Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.     

Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve-muscle cultures

Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the muscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor.

The role of basic fibroblast growth factor (bFGF) in signaling the development of the neuromuscular junction was examined. Beads coated with bFGF induced the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus myotomal muscle cells. Tyrphostin, a tyrosine kinase inhibitor, abolished AChR clustering induced by bFGF beads, suggesting a role of tyrosine kinase activation in AChR clustering. Using specific antibodies, we demonstrated the presence of both bFGF and its receptor in the myotomal muscle in vivo during the period of neuromuscular connection. However, similar tissue from older animals with mature neuromuscular junctions showed an apparently truncated form of the bFGF receptor. These data suggest that bFGF may play a role in signaling synaptogenesis in skeletal muscle.     

The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins.

In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions

We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.     

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.     

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.     

The influence of AChR clustering stimuli on the formation and maintenance of AChR clusters induced by polycation-coated beads in Xenopus muscle cells

Nerve, polycation-coated beads, and electric fields not only induce acetylcholine receptors (AChRs) to cluster, but they also reduce the number of spontaneous AChR patches (hotspots) away from the induced cluster sites on embryonic Xenopus myotomal muscle cells grown in tissue culture (the global effect). In vivo, the ability of an AChR clustering stimulus to depress cluster formation elsewhere on the muscle cell may influence both the site at which the neuromuscular junction develops as well as which axons survive during synapse elimination. Since the causes of hotspot formation may be variable and cannot be controlled, we have further characterized the global effect by using AChR-clustering stimuli that can be controlled by the experimenter. We report that innervation inhibits the formation and maintenance of bead-associated AChR patches (BARPs) by a percentage of polycation-coated beads. We next investigated competition between beads and between beads and electric fields. In competition between beads added to the muscle cells at different times, however, the first set of beads had a competitive advantage over the second set of beads. This advantage was strengthened when the latency between bead applications was extended, or when a relatively large number of BARPs were formed by the first set of beads. Likewise, long-term electric fields were able to prevent the formation of BARPs, but were unable to disperse mature BARPs. Longer electric fields, or electric fields of greater magnitude competed better with the beads than brief or weak field treatments. None of the "winning" stimuli, including nerve, were able to totally block AChR patch formation or maintenance by competing stimuli. Thus, the global effect, at least in the case of competition between nonneuronal stimuli, favors the initial stimulus and appears to be graded.