One of the earliest events in the formation of a synapse between nerve and muscle -- the neuromuscular junction -- is the aggregation of acetylcholine receptors (AChR) in the region of the muscle membrane contacted by nerve. This aggregation, or clustering, of AChR involves both intracellular and extracellular macromolecules. Similar molecules participate in AChR clustering in vitro, in muscle cultured in the absence of nerve. The AChR clusters of cultured rat myotubes can be purified greater than or equal to 100 fold with respect to cellular protein with little apparent change in organization. The cytoskeletal proteins and their association with AChR can be easily studied in such preparations. The preliminary experiments we have done suggest that AChR clusters are organized like the membrane skeleton of the human erythrocyte. This hypothesis will be carefully examined using a variety of biochemical and morphological techniques. AChR clusters will be isolated and treated in different ways to deplete the various components of the membrane skeleton selectively. Samples will then be probed with antibodies to the cluster-associated cytoskeletal proteins, and with antibodies to the cytoplasmic domains of the different subunits of AChR. Quantitative immunofluorescence techniques should reveal the identity of the receptor subunit(s) in close proximity to the membrane skeleton. Quantitative binding studies using radiolabeled antibodies and -bungarotoxin should reveal the stoichiometry of the AChR-cytoskeleton complex. The morphology of the membrane skeleton of AChR clusters should be revealed by several ultrastructural techniques (label-fracture; quick-freeze, deep-etch, rotary shadowing; negative staining). The sequence of events during disassembly and reassembly of AChR clusters will be determined by both quantitative fluorescence and ultrastructural techniques. Finally, some of the biochemical interactions involved in maintaining AChR clusters will be determined in binding and gel blotting experiments. These experiments should reveal many of the details of the organization of the AChR clusters isolated from cultured rat myotubes. As these structures are very similar to the clusters which form at early stages of formation of the neuromuscular junction in vivo, the results should help us to understand some of the molecular events involved in synaptogenesis.
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