One of the earliest events in the formation of a synapse between a motor neuron and a skeletal muscle fiber--a neuromuscular junction--is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane. This step of synapse formation is approximated by rat myotubes maintained in tissue culture, which form large aggregates or clusters of AChRs where they adhere to the substrate. The relationship between AChR clustering and adhesion of the myotube to the substrate or to other extracellular materials is found in all types of muscle cells so far examined. One may therefore pose the question, """"""""What property associated with adhesive sites promotes the nearby clustering of AChRs?"""""""". The object of the research proposed in this application is to identify the extracellular macromolecules associated with myotube-substrate adhesion, and to learn how they are related to AChR clustering. Substrate-attached material (SAM) will be prepared from partially purified AChR clusters. SAM can be studied in situ, or after solubilization. Some of the glycoproteins in SAM will be purified on con A-Sepharose, and by HPLC. Monoclonal antibodies will be generated against them. Other components of myotube SAM will be characterized using monospecific, affinity-purified antibodies generated against rat muscle adherons. Using immunofluorescence and scanning electron microscopy, the various antibodies will be used to localize their antigens in SAM. The antibodies will also be used to determine if their antigens are present at embryonic or adult neuromuscular junctions of the rat. The possibility that adherons are present at the myotube-substrate attachment site, and are sufficient to induce AChR clustering in nearby muscle membrane will be investigated. Finally, the proteins in isolated AChR clusters which anchor the intracellular cytoskeletal proteins, vinculin and talin, to the membrane regions involved in myotube-substrate attachment will be identified. These experiments should establish the nature of some of the molecules in myotube SAM, and their locations in vitro and in vivo. They should further reveal: (i) some aspects of their organization in the extracellular space; (ii) their ability to induce receptor clustering; and (iii) their possible association with the cytoskeleton. This information should be helpful in evaluating the relationship between myotube-substrate adhesion and AChR clustering.
