Genetic, transgenic, physiological and ultrastructural methods are combined to investigate the structure and function of myosin heavy chain (MHC) isoforms, myosin regulatory light chain (DMLC2), and the newly- discovered myosin rod protein (MRP) in processes of myofibril assembly, stretch activation, and cross-bridge activity in functionally specialized muscles of Drosophila. These studies examine the functions of MRP and DMLC2 as molecular tethers that regulate associations between thick and thin filaments and that are proposed to be essential components of stretch activation and tension maintenance mechanisms in vertebrate cardiac muscle and insect direct and indirect flight muscles. Transgenic and biochemical studies will investigate the function of alternative exon 11 MHC """"""""converter"""""""" domains expressed in specialized Drosophila muscles through regulated, muscle-specific alternative RNA splicing. These studies test the hypothesis that the """"""""converter"""""""" domain in the MHC head controls the rate of ADP release and head detachment during the cross-bridge cycle, making this domain of myosin a critical regulator of contractile activity in muscle. The proposed experiments involve generation of transgenic lines of Drosophila that express mutant forms of MRP, DMLC2 and MHC in indirect and direct flight muscles, using muscle-specific promoters. The assembly and function of these proteins will be examined using electron microscopy flight kinematics of intact animals, and mechanical measurements on isolated myofibrils. Complementary transgenic and biochemical studies are proposed to investigate activities of the exon 11 """"""""converter"""""""" domain of the MHC isoforms as defined by alternatively spliced exons. These findings will lead to an understanding of the regulation of contractile activity in muscles and the molecular basis of stretch activations, a property by other muscle types, particularly cardiac. These discoveries will provide a basis for understanding and treating genetic diseases of cardiac muscle and other muscle types.
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