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: l) 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 have developed a mammalian ventral horn neuron-skeletal muscle coculture system in which neurites induce redistribution of acetylcholine receptors on the myotube surface in 24 to 48 hours. The same neurite can induce the accumulation of receptors along its course and reduce the concentration of receptors where it contacts a preexisting patch of high receptor density. We are characterizing this system and will use it to investigate the role of agrin and other neuronal factors in postsynaptic receptor aggregation and to study other mechanisms of synaptogenesis. We are correlating data obtained by calcium imaging, immunocytochemistry and electron microscopy on cultured skeletal myotubes to determine the steps in the assembly of functional triads. Developing triads and peripheral couplings containing ryanodine receptors (calcium release channels) and dihydropyridine receptors are found in 3 day old myotubes. Excitation-contraction coupling can occur in cells with only a few such structures. Changes in the maximal calcium release and reuptake rate occurring in the course of development are correlated with the elaboration of the sarcoplasmic reticulum around the myofibrils. We have generated polyclonal antibodies against a protein from fetal pig brain that induces the formation of acetylcholine receptor aggregates on skeletal muscle cells in culture. The antibodies immunoprecipitate all acetylcholine receptor aggregating activity in crude fractions of brain and spinal cord extract as well as in purified preparations and recognize a 120 kilodalton protein. The antibodies are being used to further purify and characterize the protein and to determine its possible relationship with agrin.
