EXCEED THE SPACE PROVIDED. We propose a multifaceted and integrative approach to determine the function of the 'hinge' region of the myosin heavy chain protein in muscle contraction. While much effort has focused on the role of the myosin head in dictating muscle contractile properties, our novel results indicate that the hinge of the rod also is critical for muscle function. We employ Drosophila melanogaster, which is amenable to genetic, transgenic and muscle mechanical approaches. Its single myosin gene encodes alternative myosin hinge regions, one expressed in slow twitch embryonic muscles and one expressed in fast twitch and oscillatory adult muscles. We produced a transgenic line that expresses myosin with the embryonic hinge in muscles that normally express the adult hinge (indirect flight and jump muscles). Transgenic muscles assemble normal-looking myofibrils, but muscle function is severely compromised. We propose to determine if hinge function is critical at the level of the intact organism, the isolated fiber, the single myofibril, the thick filament and/or the myosin molecule, by testing the following hypotheses: 1) the myosin hinge is not important in myofibril assembly; 2) the hinge influences the mechanical properties of muscle fibers and myofibrils [in collaboration with Dr. David Maughan (U. Vermont) and Dr. Gerald Pollack (U. Washington),experts in biophysical measurements of muscle fibers and myofibrils, respectively]; 3) the hinge contributes significantly to physical properties of myosin molecules and thick filaments, specifically to differences in the shortening of isolated myosin molecules or elasticity of intact thick filaments (in collaboration with Dr. Pollack); 4) the propensity to form a coiled-coil is critical to defining the differences between alternative hinge domains; 5) hinge-specific protein interactions impart functional differences between alternativehinge regions [we will test for interaction with the thick filament protein flightin (in collaboration with Dr. Jim Vigoreaux, U. Vermont) and perform genetic suppression studies in flies]. Out integrative approach should elucidate the role of the myosin hinge in muscle function and the mechanism by which it acts. Our work is relevant to human disease since mutations in myosin can cause hypertrophic cardiomyopathy in heart muscle and central core disease in skeletal muscle. Further, since the indirect flight muscle has oscillatory and stretch-activated properties similar to human cardiac muscle, understanding myosin' involvement in generating these properties may lead to insights into human heart function. PERFORMANCE SITE ========================================Section End===========================================
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