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 contracted by nerve. Proteins of the cytoskeleton and the extracellular matrix have been implicated in this process. Little is known, however, about the organization of these proteins in the postsynaptic membrane as synaptogenesis proceeds. We will address this question by studying the formation of the neuromuscular junction in tissue culture. Cocultures of nerve and muscle cells from Xenopus laevis embryos form neuromuscular junctions that resemble the junctions of mature animals. Synaptogenesis in vitro proceeds through several distinct stages that are readily recognized when fluorescent derivatives of alpha-bungarotoxin are used to mark the postsynaptic accumulations of AChR. We will use immunofluorescence techniques: (i) to identify the extracellular and cytoskeletal proteins associated with synapses; and (ii) to determine the stage of synapse formation at which these proteins become associated with the postsynaptic region. We will use quick freeze, deep eton, rotary replication methodology at the ultrastructural level; (iii) to visualize the macromolecular arrays (e.g., microfilaments, coated membrane, extracellular matrix) that are present at the postsynaptic membrane at different stages of synaptogenesis; and (iv) to localize extracellular and cytoskeletal proteins to these synaptic structures by immunogold labeling. These studies should identify the proteins that are used to assemble the postsynaptic membrane of the neuromuscular junction, localize them to particular postsynaptic structures, and elucidate the sequence of assembly of these structures during synaptogenesis. These are essential steps if we are eventually to understand synapse formation at the molecular level.
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