Synapses are highly specialized intercellular contacts having specific proteins confined to the region of contact in the plasma membrane, subjacent cytoplasm, and extracellular space. How these proteins contribute to the synaptic architecture is not yet precisely determined, nor is it understood how this complex structure is assembled beginning with an undifferentiated contact between pre and postsynaptic cells. Clusters of acetylcholine receptors (AChR), formed on the ventral membranes of rat myotubes in tissue culture, are a model system for the postsynaptic membrane of neuromuscular junctions. These clusters are made up of interdigitating domains that are either rich in AChR and its associated proteins, or are attached to the substrate with concomitant links to a cytoskeleton composed of bundled actin microfilaments or polymerized clathrin. Clusters are isolated by mechanical shearing and identified by binding of fluorescent toxin to AChR. Freeze fracture and quick-freeze, deep-etch replication reveals characteristic supramolecular assemblies of the cytoskeleton in the three domains. Since shearing removes most of the cell cytoplasm, the cytoplasmic membrane surface is readily accessible for immunolabeling with antibodies absorbed to colloidal gold. Separating the replicated membrane from its supporting coverslip exposes its external surface for immunolabeling and replication. Labeling techniques will determine the relative locations of particular proteins contributing to each domain. Emphasis will be on the cytoskeleton and basal lamina proteins immobilizing AChR, and on the organization of proteins responsible for filament and substrate attachment at contact domains of myotubes and focal contacts of fibroblasts. A sequence of steps was previously proposed for cluster formation, including formation of contact domains followed by clathrin-coated domains and finally by insertion of AChR and assembly of AChR domains. Observations on clusters at various stages of development will be used to verify or disprove this scheme.
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