The major objective of this project is to use Drosophila mutants to study the role of the myosin """"""""hinge"""""""" region in muscle structure and contraction. The Drosophila myosin heavy chain gene is unique in that a single copy of the gene encodes all muscle myosin heavy chains through alternative splicing, and mutants affecting muscle function can be easily isolated and analyzed. Genes mutated in vitro can be stably inserted into the germline. Furthermore, expression of mutant forms of the protein in certain muscles (flight, and jump) can be studied without concern that altered function might cause lethal phenotypes. The project will focus on the study of phenotypes of these muscles when their myosins contain an alternative hinge substituted for the wild type. The alternative hinge is normally expressed in muscles with very different mechanical and ultrastructural properties than flight and jump muscles. We will determine the effects of this substitution on flight and jump ability of adult organisms. We will study whether changes in myofibril ultrastructure are brought about by changes in the myosin hinge. We will use antibodies against hinge peptides to determine whether there is differential localization of myosins with alternative hinges in muscles that express myosins having both types of hinge. Using similar approaches to those outlined above, we will examine whether alternative C-terminal """"""""tailpieces"""""""" of the myosin rod affect structural and functional characteristics of Drosophila muscle. If altered function is detected in the organisms that have switched hinge or tailpiece regions, future studies will involve analysis of: l) point mutants designed to test critical amino acid residues imparting altered function, 2) mechanical properties of dissected flight and jump muscle myofibers and single isolated myofibrils, and 3) in vitro motility where mechanical interaction of proteins can be studied directly. In addition, genetic suppression analysis will be used to determine regions of myosin and other proteins that interact with the myosin hinge. Overall, by using a molecular genetic approach coupled to structural and functional assays, we will directly determine the impact of alternative regions of the myosin rod on muscle physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR043396-03
Application #
2683330
Study Section
Physiology Study Section (PHY)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
2000-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
San Diego State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
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
Hao, Yudong; Bernstein, Sanford I; Pollack, Gerald H (2004) Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy measurement method. J Muscle Res Cell Motil 25:359-66
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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