This project is an outgrowth of work in this laboratory using ultrastructural methods and immunocytochemistry to study cytoplasmic proteins associated with the postsynaptic membrane of Torpedo electric tissue. The main long-term goal has been, and remains, to identify the mechanisms by which accumulations (""""""""clusters"""""""") of acetylcholine receptor molecules are stabilized (""""""""anchored"""""""") against lateral diffusion in the plasma membrane. In this proposal, the underlying idea--that anchoring is mediated by cytoskeletal and other cytoplasmic structures--is expanded to include a role for cluster-specific basal lamina, and the work is shifted to skeletal muscle from rat and Xenopus laevis. The previous methods plus freeze-fracture/deep-etch electron microscopy will be applied to clusters on muscle cells in culture, developing neuromuscular junctions, and the mature junction. Cytoskeletal, other cytoplasmic, and extracellular proteins at clusters will be identified immunocytochemically and localized relative to the receptor, the structures in which these proteins occur will be visualized in 3-dimensions, and how identified proteins fit into these structures will be determined. It is hoped that by judicious choices of sample and with the aid of cell biological manipulation of clusters, the various roles of these structures in receptor anchoring, in maintenance of the cell-cell contact nature of the junction, and in other possible synaptic activities can be delineated.
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