Synapses are highly specialized cell-cell contacts having specific proteins confined to the region of contact in the plasma membrane, subjacent cytoplasm, and extracellular space. Receptor clusters formed by rat myotubes in tissue culture are a model system for the postsynaptic membrane of neuromuscular junctions. These clusters combine a high concentration of a specific postsynaptic protein, the acetylcholine receptor (AChR) with an enhanced attachment to the underlying substrate. Additional proteins common to AChR clusters and neuromuscular junctions have been identified by immunofluorescence. Interference reflection microscopy and localization of AChR by fluorescence and freeze fracture have shown that receptor clusters are composed of interdigitating domains either enriched in AChR or lying especially close to the substrate. These clusters may be isolated by detergent extraction or mechanical shearing, then prepared for electron microscopy by freeze fracture or rapid-freeze, deep-etch, rotary-shadowing. Since most of the cell cytoplasm is removed by shearing, the cytoplasmic surface of the membrane is readily accessible for antibody labeling. Several proteins have been identified in postsynaptic membranes and AChR clusters by immunofluorescence; antibodies to these proteins will be used to label replicas using second antibodies adsorbed to colloidal gold. Localization studies should help to clarify the proteins involved in attachment of microfilament bundles to the external substrate, the spatial relation of AChR to these attachment regions, and the intracellular network which holds AChR molecules in position within AChR-rich domains.
Showing the most recent 10 out of 18 publications
