Myosin II is responsible for the powering of skeletal and cardiac muscle as well as cell locomotion and cytokinesis. Defects in myosin function have been shown to cause hypertrophic cardiomyopathy in humans. The long term goal of the proposed studies is to understand how this critical molecular motor converts the energy of ATP hydrolysis into force and how that force generating machinery is organized within cells and tissues to produce directed cell locomotion and cytokinesis. There are three specific aims proposed in this application. (1) To use biochemical and structural analysis together with measurements of the kinetic and mechanical properties of mutant myosin molecules and to produce novel mutations to test specific hypotheses regarding the role of light chains in conversion of chemical energy into force generation. (2) To distinguish between alternative models for myosin force generation during cell locomotion by following the dynamics on myosin reorganization during locomotion and changes in cell direction. (3) To define the mechanisms responsible for regulating myosin during cell migration and cytokinesis. Specifically we will undertake a direct test of the role of myosin phosphorylation in cultured mammalian cells using gene replacement and transgene expression. We will also employ a novel biosensor to monitor the state of MLCK activity and localization as a means of exploring the mechanism by which MLCK regulates myosin activity within the context of migrating cells. The primary experimental approaches used in these studies include the molecular genetic manipulation of myosin light chains and the characterization of the consequences of these manipulations on the biochemistry, cellular localization and phenotypes of the cells, tissues and organisms bearing these mutations. These studies will increase our 0understanding of the fundamental mechanisms by which cells move and how the molecular motor myosin contributes to cell movement. Cell movement is critical for normal embryonic development, for normal physiological processes such as wound healing, the immune response to infection and angiogenesis. Analysis of these mutant light chains may also provide insights into how light chain mutations in ventricular light chain isoforms might contribute to cardiac hypertrophy.
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