My research is aimed at understanding how a synapse forms. The synapse I am studying is that formed between the motor neuron and skeletal muscle in vertebrates -- the neuromuscular junction. I have been concentrating on is the postsynaptic membrane, where acetylcholine receptors (AChR) accumulate, or cluster. The questions I am trying to answer are: What influence does the nerve exert on the muscle to cause AChR to accumulate postsynaptically? And what mechanisms does the muscle use to form and maintain the AChR aggregate? Most of the research I have been doing has focussed on AChR clusters which form in cultured rat myotubes in the absence of nerve. These clusters, which form preferentially where the myotube attaches to the substratum, can be partially purified, and studied by both morphological and biochemical means. To answer the first question, I have been investigating the extracellular macromolecules associated with AChR clusters. Some of these molecules are localized at the sites of myotube-substrate contact within AChR clusters. My working hypothesis is that they may halp to mediate muscle-to-nerve contact in the developing rat embryo. These molecules have been isolated from clusters by selective detergent extraction. Some of the components are now being characterized immunologically. Others are being purified by affinity chromatography on lectin columns. By characterizing these molecules, localizing them at developing synapses, and, ultimately, using blocking antibodies against them to try to inhibit synaptogenesis, I should be able to test by hypothesis. To answer the second question, I have been characterizing the cytoskeletal components of the AChR clusters. I have found that actin, spectrin and the 43K protein are associated with AChR clusters, and that when one or more are selectively removed, the organization of AChR clusters changes. The stoichiometry of this """"""""complex"""""""" appears to be 1 AChR: 1 spectrin: several actin monomers. These proteins may be organized like the human erythrocyte membrane-cytoskeleton. Experiments to test this hypothesis at the biochemical level are now in progress.
