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.     

Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures

We have investigated the sequential changes of acetylcholine receptor (AChR) distribution on identified Xenopus laevis muscle cells in culture before and after innervation. AChRs on muscle cells were stained with tetramethylrhodamine-conjugated alpha-bungarotoxin and the distribution of AChR clusters was examined on a fluorescence microscope using an image intensifier. Large receptor clusters were identified on muscle cells and their fate was followed afterward. In muscle cells cultured without neural tube cells, about one-half of the identified AChR clusters survived for 2 days. In nerve-muscle cocultures, preexisting AChR clusters survived longer on non-nerve-contacted muscle cells than on muscle cells cultured without nerve. However, in nerve-contacted muscle cells the great majority of preexisting AChR clusters dispersed within 2 days. The dispersal of preexisting AChR clusters preceded receptor accumulation along the path of nerve contact by about 12-16 hr. Therefore, an accelerated dispersal of receptor clusters in innervated muscle cells is not a consequence of receptor accumulation along the nerve. The preexisting AChR clusters located near and far from the nerve contact sites dispersed along a similar time course. Protease inhibitors, trasylol and leupeptin, reduced the nerve-induced dispersal of the preexisting AChR clusters in the period before AChR accumulation at the nerve contact sites but did not do so during the period when AChRs began to accumulate at nerve-muscle contact. The significance of the dispersal of preexisting receptor clusters is discussed with regard to neuromuscular junction formation.     

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.     

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.     

The effects of electrical inactivity and denervation on the distribution of acetylcholine receptors in developing rat muscle

Explants of thoracic body wall from rat embryos, including intercostal muscles, ribs, and the adjacent segments of spinal cord, were maintained in organ culture. Nerve-muscle differentiation proceeded in culture with a pattern and time course similar to that of the same synapses developing in utero. To understand further the factors that regulate acetylcholine sensitivity in developing rat myotubes, we studied the effects of electrical inactivity and denervation on the distribution of acetylcholine receptors. When muscle and spinal cord were explanted at 15 days of gestation, prior to the appearance of junctional receptor clusters, intact nerve terminals were required to initiate receptor aggregation at the site of nerve-muscle junction. Electrical activity was not necessary for induction of these primary junctional clusters. Inactivity resulted, however, in the appearance of secondary multiple receptor clusters at random sites along the fibers. In the presence of tetrodotoxin, the electrically inactive nerve terminals sprouted; this was accompanied by the enlargement of the junctional receptor clusters, at the end plate, but there was no correlation between nerve sprouting and the location of extrajunctional receptor aggregates. Later in development, at a time when the junctional receptors are metabolically more stable, terminal sprouting failed to induce the increase in size of junctional receptor aggregates.     

Neural influence on the postnatal changes in acetylcholine receptor distribution at nerve-muscle junctions in the mouse

The effect of denervation at different stages of development on the pattern of junctional AChR molecules has been examined in hindlimb muscles of the mouse using fluorescent α-bungarotoxin. Denervation at birth leads in a few days to a marked dispersal of the junctional AChR cluster. The mechanism of dispersal is unknown but appears to be too slow to be explained by free diffusion of individual AChR molecules. Following birth, the morphological stability of the cluster increases so that denervation at 2 weeks of age, when the mature form of the cluster begins to develop, leads to little increase in cluster size. The changes in the pattern of receptors seen when the nerve is intact are arrested by denervation.     

Human myotube differentiation in vitro in different culture conditions

Human muscle cells derived from satellite cells, maintained in standard tissue culture conditions, do not differentiate as rapidly or as completely as myoblasts from other species (chicken, rat, mouse). In an attempt to improve myogenesis, we studied the effects of modifying the culture media and of coculturing muscle with nerve cells, using myoblasts grown in standard culture media as the basis for comparison. Myogenesis was measured by fusion index, creatine kinase (CK) activity; acetylcholinesterase (AChE) activity (total and molecular forms); and the number of acetylcholine receptors (AChR). Modification of culture media accelerated fusion of myoblasts, but the cell density decreased and myotubes were unable to survive for long periods. In contrast, coculturing muscle with nerve cells increased both cell density and the number of myotubes. CK, AChE and AChR increased in the presence of defined media. In the nerve-muscle cocultures the increase was less marked. Manipulating culture conditions modified the molecular forms of AChE. Only a (4 + 6.5) S peak was present in control cultures, but a 10S peak appeared in defined media. The 16S form was detected only in nerve-muscle cocultures. This study shows that fusion of human myoblasts and differentiation of myotubes in tissue culture can be accelerated by removal of serum macromolecules. Further differentiation of myotubes was achieved only in the nerve-muscle cocultures.     

Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken.

Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes. At day 19 extrajunctional AChR mRNA clusters have disappeared and myogenin mRNAs are no longer detected in PLD muscle. Moreover, both myogenin and CMD1 mRNA levels increase after muscle denervation in chicks. These data are compatible with a role for myogenic factors in the induction and maintenance of extra-junctional expression of the AChR genes during early muscle development. Using digoxygenin labelled RNA probes, we also show that the mRNAs for the AChR alpha-subunit display a punctated, probably perinuclear distribution, whereas mRNAs for myogenic genes accumulate in the sarcoplasm around subsets of nuclei in the muscle fiber.     

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.     

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.     

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.     

Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures

At the adult neuromuscular junction, acetylcholine (ACh) receptors are highly localized at the subsynaptic membrane, whereas, embryonic myotubes before innervation have receptors distributed over the entire surface. Thus sometime during development, ACh receptors accumulate to the nerve contact area. This nerve-induced receptor accumulation can be reproduced in Xenopus nerve-muscle cultures, which provides us with a unique opportunity to investigate the underlying molecular mechanism of this event. Anderson and Cohen (1977) have shown that nerve-induced receptor accumulation is, at least partly, due to migration of pre-existing receptors. It is, thus, plausible that freely diffusing receptors in the membrane are trapped at the nerve-contact region and form clusters. We tested this diffusion trap model. First, receptors in the background region are indeed predominantly mobile and those in the cluster are immobile. Second, the diffusion of receptors in the membrane is fast enough to account for the rate of receptor accumulation. Third, when receptors were immobilized by a lectin, Concanavalin A, the nerve no longer induced receptor accumulation. Thus the diffusion trap model seems adequate to accommodate these observations. Aside from this diffusion mediated mechanism, it is conceivable that newly formed receptors are preferentially inserted at the nerve contact site and these new receptors become immobilized at the site of insertion. To test this hypothesis we stained new receptors separately from old ones and quantitatively compared their distribution. For this purpose we developed a method to quantify fluorescence micrographs. We found that the ratio between old and new receptors was similar at all nerve-induced clusters examined and at the diffusely distributed region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implication of nNOS in the enlargement of AChR aggregates but not the initial aggregate formation in a novel coculture model

The aggregation of nicotinic acetylcholine receptors (AChRs) is an early hallmark of the formation of neuromuscular junction (NMJ), and nitric oxide is recently known to play an important role. In many NMJ studies, nerve-muscle coculture model was used, and NG108-15 cells, a neuroblastoma x glioma hybrid cell line, were the most frequently used nerve cells. However, possible contributions from glial cells could not be excluded. In this study, Neuro-2a neuroblastoma cells were used instead of [corrected] coculture with myotubes, and the relationship between AChR aggregation and spatiotemporal expression and activation of nNOS (neuronal nitric oxide synthase) was examined. Upon coculture, AChR aggregates were observed by FITC-conjugated alpha-bungarotoxin, and double labeling of AChRs and neurofilament showed that the neurites of a Neuro-2a cell innervated several myotubes. After treating the cocultures with single dose of L-NAME at the end of 1-day [corrected] coculturing, only slight effect on AChR aggregation could be found indicating that nNOS is not related to the initial formation of AChR aggregates. In contrast, when L-NAME treatment was given at the end of a 3-day coculturing, the day just before reaching the maximum extent of AChR aggregation, new AChR aggregates were hardly formed and the preformed AChR aggregates were even dispersed indicating that the enlargement of AChR aggregates is highly dependent on the nNOS activity. Double-labeling study of nNOS and AChR further showed that the coupling of membranous nNOS to regions nearby the AChR aggregates was essential for the enlargement of AChR aggregates. These results not only revealed the spatiotemporal relationship between AChR aggregation and nNOS activity but also verified the feasibility and usefulness of using Neuro-2a cells in a coculture model.     

Cell culture-based analysis of postsynaptic membrane assembly in muscle cells

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.     

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.     

Denervation alters the size, number, and distribution of clusters of acetylcholine receptor-like molecules on frog cardiac ganglion neurons

Acetylcholine receptor (AChR)-like molecules are found in clusters on the surface of parasympathetic neurons in the frog cardiac ganglion. Electron microscopy of immunoperoxidase-stained tissue reveals that in normally innervated ganglia most of these clusters are located at synaptic sites. Denervation for 2-3 weeks results in a 64% reduction in the total surface area occupied by AChR-like clusters; this change is brought about by the combined effects of a 4-fold decrease in cluster size and a 30% increase in cluster number. Denervation also changes the distribution of AChR-like clusters: clusters, normally restricted to portions of the cell surface, are more widely distributed following denervation. Denervation of amphibian skeletal muscle for a comparable period of time has no effect on the size or the number of synaptic clusters of AChRs. These results suggest that AChRs in nerve and in muscle are regulated differently by innervation.     

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.     

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.     

Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.     

Actin at receptor-rich domains of isolated acetylcholine receptor clusters

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR-rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin-like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.