We carry on basic research into the organization of the myofibrils and excitable membrane systems 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. Immunocytochemical studies on isolated myofibrils from rat and chicken skeletal muscle have shown that tropomodulin, a novel tropomyosin-binding protein, is associated with the ends of actin thin filaments at a stoichiometry of 1-2 tropomodulin molecules per filament. It is suggested that tropomodulin has a role in regulating thin filament length and/or is involved in crosslinking the thin filament ends. 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, containing ryanodine receptors (calcium release channels) and dihydropyridine receptors are concentrated around nuclei in 3 day old myotubes. The role of these developing triads in calcium release is being examined. The development of rhythmic spontaneous contractions and the corresponding calcium transients occurs later and is correlated with the appearance of triads throughout the myotube. We have generated monoclonal and polyclonal antibodies against a protein from fetal pig brain that induces the formation of acetylcholine receptor aggregates on skeletal muscle cells in culture. The polyclonal antibody immunoprecipitates all acetylcholine receptor aggregating activity in crude fractions of brain extract. The antibodies are being used to further purify and characterize the protein and to determine its histological distribution.
