We carry on basic research into the organization of the excitable membranes and myofibrils of striated muscle and the cellular and molecular mechanisms involved in their differentiation. We focus on two membrane systems in striated muscle cells: 1) The postsynaptic membrane of the skeletal neuromuscular junction 2) The membranes of the excitation-contraction coupling system, specifically the transverse tubules and sarcoplasmic reticulum that form the triad junction. We developed a mammalian spinal cord neuron-skeletal muscle culture system in which neurites induce postsynaptic-like accumulation of acetylcholine receptors on the myotubes, from as early as 6 hours of coculture. We have shown that this inductive ability is predominantly a property of developing axons, as opposed to dendrites, and that the axon- myotube contacts can develop accumulations of synaptic vesicles, a typical synaptic cleft and a differentiated postsynaptic cell surface, as in the neuromuscular junction developing in vivo. We have devised a procedure to label the neurons in the cocultures with a fluorescent membrane dye in order to simultaneously observe the interactions betwen axons and myotubes and the resulting accumulation of acetylcholine receptors. We will use this culture system to investigate the role of agrin and other putative signals in postsynaptic receptor aggregation and to study other mechanisms of synaptogenesis. We have been correlating data obtained by calcium imaging, immunocytochemistry and electron microscopy on cultured skeletal myotubes to determine the steps in the assembly of functional triads. Our results suggest that the structural and molecular differentiation of the membrane domains of transverse tubules and sarcoplasmic reticulum involved in excitation-contraction coupling occurs in concert with the formation of functional junctions between the two membrane systems. While the initial formation of these junctions is independent of association with myofibrils, we find that reorganization into proper triads occurs as the junctions become associated with the A-I border of the sarcomere.
