Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering

The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.     

Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

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

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

Recombinant neuromuscular synapses

The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.     

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

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

Lipid domains of acetylcholine receptor clusters detected with saponin and filipin

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

The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes

We have examined the relationship of acetylcholine receptors (AChR) to the Mr 43,000 receptor-associated protein (43K) in the AChR clusters of cultured rat myotubes. Indirect immunofluorescence revealed that the 43K protein was concentrated at the AChR domains of the receptor clusters in intact rat myotubes, in myotube fragments, and in clusters that had been purified approximately 100-fold by extraction with saponin. The association of the 43K protein with clustered AChR was not affected by buffers of high or low ionic strength, by alkaline pHs up to 10, or by chymotrypsin at 10 micrograms/ml. However, the 43K protein was removed from clusters with lithium diiodosalicylate or at alkaline pH (greater than 10). Upon extraction of 43K, several changes were observed in the AChR population. Receptors redistributed in the plane of the muscle membrane in alkali-extracted samples. The number of binding sites accessible to an anti-AChR monoclonal antibody directed against cytoplasmic epitopes (88B) doubled. Receptors became more susceptible to digestion by chymotrypsin, which destroyed the binding sites for the 88B antibody only after 43K was extracted. These results suggest that in isolated AChR clusters the 43K protein covers part of the cytoplasmic domain of AChR and may contribute to the unique distribution of this membrane protein.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

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.     

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

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

Association of acetylcholine receptors with peripheral membrane proteins: evidence from antibody-induced coaggregation

Acetylcholine receptors (AChR) are associated with several peripheral membrane proteins that are concentrated on the cytoplasmic face of the plasma membrane at the neuromuscular junction, and at aggregates of AChR that form in vitro. We tested the linkage among these proteins by inducing microaggregation of AChR, then determining if a given peripheral membrane protein accumulated with the receptors in microaggregates. In most experiments, we used isolated membrane fragments that are rich in AChR and accessible to antibodies against intracellular antigens. We showed that the 43 kD receptor-associated protein always aggregated with AChR, whether microaggregation was driven by antibodies to the 43 kD protein, or to the receptor itself. Antibodies to the 58 kD receptor-associated protein also always aggregated the 58 kD protein with the receptor. Our results are consistent with a model for AChR-rich membrane in which the 43 kD and 58 kD proteins are both closely associated with the AChR.When we induced microaggregation in intact muscle cells with anti-AChR antibodies, our results were less definitive. The 43 kD receptor-associated protein microaggregated with AChR, but the 58 kD protein was not especially enriched at AChR microaggregates. We discuss the advantages of using isolated AChR-rich membrane fragments to study the association of AChR with peripheral membrane proteins.We thank J. Strong for his expertise in obtaining the data for Figs. 6–8, and W. Resneck, A. O'Neill, and K. Douville for their assistance throughout this work. Our research has been supported by grants from the Muscular Dystrophy Association (to R.J.B., R.S., D.W.P. and S.C.F.) and from the National Institutes of Health (NS17282 to R.J.B.; NS27171 to P.W.L.; NS15513 to D.W.P.; NS15293 to R.S.; NS14871 to S.C.F.).     

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

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

Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes

Two populations of acetylcholine receptors (AChRs) are present in cultured myotubes. One forms large aggregates or clusters and the other has a much lower density of AChRs, which are diffusely distributed. Both clustered and diffuse AChRs are inserted and removed (internalized) from the sarcolemma. To determine the insertion and removal rates of AChRs in these two plasma membrane domains, we used a double label technique to distinguish and quantitate newly inserted and "old" AChRs. Application of our method revealed that the rate of AChR internalization is the same at the clustered and diffuse regions of the plasma membrane, whereas the rate of insertion is threefold greater at the clusters than elsewhere in the plasma membrane. Thus, the increase in AChR number at the clusters is not due to an increase in their half-life, but to an increase in their rate of insertion.     

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

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

Increased expression of the 43-kD protein disrupts acetylcholine receptor clustering in myotubes

The 43-kD protein is a peripheral membrane protein that is in approximately 1:1 stoichiometry with the acetylcholine receptor (AChR) in vertebrate muscle cells and colocalizes with it in the postsynaptic membrane. To investigate the role of the 43-kD protein in AChR clustering, we have isolated C2 muscle cell lines in which some cells overexpress the 43-kD protein. We find that myotubes with increased levels of the 43-kD protein have small AChR clusters and that those with the highest levels of expression have a drastically reduced number of clusters. Our results suggest that the 1:1 stoichiometry of AChR and 43-kD protein found in muscle cells is important for AChR cluster formation.     

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

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

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.     

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

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

Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells.

Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules on living, nondeoxygenated cells with characteristic times previously ranging from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rotating membrane protein monomers that typically should have a characteristic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With this new system, PFRAP experiments can be conducted with sample time as short as 0.4 microseconds and detection of possible characteristic times of less than 2 microseconds. The system is tested on rhodamine-alpha-bungarotoxin-labeled acetylcholine receptors (AChRs) on myotubes grown in primary cultures of embryonic rat muscle, in both endogenously clustered and nonclustered regions of AChR distribution. It is found that approximately 40% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian motion. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregating factors) on the myotube AChR were also examined by the fast PFRAP system. Brain extract is known to abolish the presence of endogenous clusters and to induce the formation of new clusters. It is found here that rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotational diffusion in the nonclustered regions of extract-treated myotubes remains rapid.