Myosin II is the motor molecule responsible for powering the contraction of skeletal and cardiac muscle, as well as contraction of the contractile ring during cytokinesis in virtually all cells. Defects in myosin II function are responsible for human diseases such as hypertrophic cardiomyopathy. Despite nearly 50 years of in vitro biochemical study, it has only recently become possible to correlate the wealth of biochemical information with in vivo function. Dictyostelium has been an important system which has made many of the in vivo studies possible. Myosin II molecules have three domains, an alpha-helical coiled coil that leads to formation of thick filaments through side-to-side associations, a head domain that contains the active site of the enzyme as well as the actin binding site, and a neck or regulatory domain that links the heads to the rod domain and carries both types of light chains. The motor activity of myosin is driven by ATP hydrolysis, which is thought to produce conformational changes in the structure of the myosin head which generates movement of the myosin head relative to an actin filament. The myosin molecule is a hexamer consisting of two copies each of the myosin heavy chain (MHC), two copies of the essential light chain (EMLC) and two copies of the regulation light chain (RMLC). In non-muscle cells and smooth muscle the activity of myosin is regulated by phosphorylation of serine residues on the RMLC. Dictyostelium cell lines in which the EMLC or RMLC gene have been disrupted by gene targeting have established that both the EMLC and RMLC are required for normal myosin function. The long term goal of the proposed research is to understand the mechanisms of cell motility and specifically how the MLCs contribute to motility. The specific goals of the proposed experiments are to; (1) further characterize the phenotypic and biochemical defects of cells lacking MLC expression or expressing mutant MLCs, (2) to determine what essential functions that EMLC and RMLC provide to the myosin molecule, and (3) to investigate the contribution of cell motility to the morphogenetic movements that occur during tissue development using Dictyostelium as a model. These experiments should contribute to our understanding of how myosin, a critical component of virtually every cell functions, contributes to cell motility and how cell movement contributes to tissue formation. Since myosin has been implicated in human disease, improved understanding of these fundamental mechanisms may contribute to improved diagnosis and treatment of disease processes involving contractility and cell motility such as hypertrophic cardiomyopathy, metastasis and birth defects.
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