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.
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