The overall aim of the research proposed here is to understand the process of synaptogenesis at the molecular level. The synapse to be studied is the neuromuscular junction (NMJ) of vertebrates. The NMJ has many distinctive features, including a paracrystalline array of acetylcholine receptors (AChR) in the postsynaptic membrane (PSM), a characteristic postsynaptic density, and a specialized basal lamina. Experiments are proposed to elucidate some of the mechanisms responsible for the development of these synaptic specializations. An early step in the formation of the NMJ is the clustering of AChR at the PSM. AChR clusters also form in aneural cultures of rat myotubes. These clusters, which form preferentially where the myotube adheres to the tissue culture substrate, are very similar to the clusters that form at the embryonic NMJ in response to innervation. They are organized into 3 distinct, interdigitating membrane-cytoskeletal domains, one highly enriched in AChR, and two devoted to cell-substrate attachment. The AChR- rich domain contains a network of an unusual isoform of beta-spectrin that is probably linked to the AChR by the 43K and 58K peripheral membrane proteins. AChR-rich domains have a distinctive extracellular matrix (ECM) that may be stabilized by interacting with the cytoskeleton. Some of the questions addressed here are: What is the molecular nature of the spectrin associated with AChR? How is it linked to the AChR? What other components are present in the network, and how are they bound there? Which of the proteins in the network is responsible for stabilizing the ECM? These questions will be addressed using immunological and morphological techniques. In addition, molecular cloning experiments are proposed to characterize the receptor-associated form of spectrin and to compare it to more common isoforms of this protein. The two other domains of AChR clusters, responsible for cell-substrate adhesion, are focal contacts and large areas of coated membrane. The similarities of AChR clusters in vitro to the aggregates of AChR that form in vivo at NMJs of embryonic rats suggest that the early PSM may also be organized into domains. Morphological studies of the embryonic PSM show clear AChR-rich and AChR-poor areas. Are these areas organized like the AChR-rich and AChR-poor domains of AChR clusters in vitro? If so, how ar they modified as the NMJ matures? Confocal immunofluorescence studies are proposed to answer these questions by examining the distribution of proteins at the embryonic, neonatal and adult NMJ. How chemical synapses form during embryogenesis and early postnatal development is still only poorly understood. Elucidating the organization of the PSM of the embryonic NMJ, and how it changes with development, should contribute to an understanding of this process. Ultimately, what is learned about the NMJ should help to explain how chemical synapses form throughout the developing nervous system, and how synaptogenesis can be altered by disease, by drugs, or by genetic factors.
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