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===========================================

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Cell Development and Function Integrated Review Group (CDF)
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Nuckolls, Glen H
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San Diego State University
Schools of Arts and Sciences
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Cammarato, Anthony; Li, Xiaochuan Edward; Reedy, Mary C et al. (2011) Structural basis for myopathic defects engendered by alterations in the myosin rod. J Mol Biol 414:477-84
Miller, Mark S; Dambacher, Corey M; Knowles, Aileen F et al. (2009) Alternative S2 hinge regions of the myosin rod affect myofibrillar structure and myosin kinetics. Biophys J 96:4132-43
Cammarato, Anthony; Dambacher, Corey M; Knowles, Aileen F et al. (2008) Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles. Mol Biol Cell 19:553-62
Suggs, Jennifer A; Cammarato, Anthony; Kronert, William A et al. (2007) Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length. J Mol Biol 367:1312-29
Hao, Yudong; Miller, Mark S; Swank, Douglas M et al. (2006) Passive stiffness in Drosophila indirect flight muscle reduced by disrupting paramyosin phosphorylation, but not by embryonic myosin S2 hinge substitution. Biophys J 91:4500-6
Melkani, Girish C; Cammarato, Anthony; Bernstein, Sanford I (2006) alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress. J Mol Biol 358:635-45
Liu, Hongjun; Miller, Mark S; Swank, Douglas M et al. (2005) Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster. Proc Natl Acad Sci U S A 102:10522-7
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Liu, Hongjun; Mardahl-Dumesnil, Michelle; Sweeney, Sean T et al. (2003) Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation. J Cell Biol 160:899-908
Zhang, S; Bernstein, S I (2001) Spatially and temporally regulated expression of myosin heavy chain alternative exons during Drosophila embryogenesis. Mech Dev 101:35-45

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