We will use an integrative and multidisciplinary approach to investigate how the head domain of the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. We will test a series of basic and novel hypotheses that predict biochemical, fiber mechanical and Iocomotory properties imparted by specific myosin domains. An innovative aspect of our system is that the functions of individual domains and residues will be tested in vitro, in muscle cells and in intact organisms. Therefore, we can determine directly and to what degree a specific biochemical property defines a mechanical or Iocomotory characteristic. To this end, we will employ a battery of in vitro and in vivo assays: ATPase, actin and nucleotide affinity, in vitro motility, molecule image reconstruction, electron microscopy, isolated fiber mechanics and organismal locomotion. Our studies use the model organism Drosophila melanogaster because it has a single muscle Mhc gene, but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, we create "isoform-switch" organisms that accumulate chimeric (Aim 1) or naturally occurring (Aim 2) versions of MHC differing in single alternative head domains. By integratively analyzing these transgenic organisms we will validate or refine hypotheses regarding myosin domain function at multiple levels. We will then produced transgenic organisms to define the function of the N-terminal beta-barrel domain and to elucidate the tuning mechanism of the relay loop (Aim 3). Finally, we will combine the transgenic approach with classical genetics to introduce and suppress a mutation in an amino acid residue hypothesized to be critical for communication between the myosin converter and relay loop domains (Aim 4). By interpreting our results in relation to the three-dimensional structure of myosin, we will help define the molecular interactions necessary for muscle contraction. Overall, our novel integrative analyses will permit testing of models for the transduction of chemical energy into movement and will yield direct insight into how myosin functions in muscle. Since mutations in the myosin head cause defects in human cardiac and skeletal muscle, these studies are relevant to understanding human myopathies.
NIH ExPorter
NIH ExPorter