Synapses are highly specialized intercellular contacts. Specific proteins of the plasma membrane, subjacent cytoplasm, and extracellular space are confined to the region of contact. 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, These questions are addresses with a model system for the postsynaptic membrane, the clusters of acetylcholine receptors (AChR) that form on the ventral membranes of rat myotubes in tissue culture. Clusters are identified by binding of fluorescent toxin to AChR and isolated by mechanical shearing. The cytoplasmic membrane surface and its adherent membrane skeleton are revealed by quick-freeze, deep-etch and platinum replication (QFDERR), and protein component are localized by immunogold labeling. The proposed research concentrates on AChR-rich domains of clusters, whose membrane skeleton is composed primarily of an isoform of beta-spectrin organized into a network of intersecting filaments similar to that found in the membrane skeleton of erythrocytes.
In specific aim 1, immunogold labeling will be used to determine the positions of AChR and its related proteins relative to intersections in the membrane skeletal network and to suggest how the network is linked to the integral membrane AChR.
In specific aim 2, QFDERR and immunogold labeling will be used to study AChR- associated proteins, and mutated variants of these proteins, expressed in non- muscle cells. These experiments should further elucidate the role of these proteins, and particular portions of their sequence, in aggregation of AChR and formation/attachment of a membrane skeleton. They also open the way to further molecular biological studies of the architecture, formation, and maintenance of spectrin-based membrane skeletons in other less-accessible tissues.
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