Our goal is to study the cellular and molecular mechanisms involved in the assembly, remodeling and elimination of chemical synapses. Our primary approach is to study the formation, stabilization and elimination of nicotinic acetylcholine receptor aggregates on striated embryonic muscle fibers grown in tissue culture. The formation of these receptor aggregates is induced by neuronal factors present in embryonic brain extract. The acetylcholine receptor aggregates, together with associated cell surface structures, provide a model of postsynaptic differentiation at the skeletal neuromuscular junction. We use fluorescence and electron microscopy, as well as immunocytochemistry to follow changes in the distribution of acetylcholine receptors and associated cell surface components, and to study the underlying ultrastructural changes. In the past year, we have made the following observations: 1. Newly formed acetylcholine receptor aggregates contain at least two distinct types of membrane-cytoskeletal domains: a) receptor enriched domains, associated with a 43 kilodalton protein and actin. b) """"""""adhesion"""""""" domains, associated with vinculin, and actin filament bundles. 2. Newly formed acetylcholine receptor aggregates also contain punctate sites of clathrin immunoreactivity, possibly representing coated pits. 3. As seen by immunogold labeling, the 43 kilodalton protein is precisely colocalized with acetylcholine receptors at the crests of the postsynaptic folds in the neuromuscular junction, and in acetylcholine receptor aggregates formed in culture. In contrast, sodium channels are distributed throughout the postsynaptic membrane folds of the neuromuscular junction, at an apparently lower site density than the acetylcholine receptors at the crests.
