Myosins are biological motor proteins which translocate actin filaments upon hydrolysis of ATP. It has become clear that myosins constitute a superfamily of proteins which contain a conserved motor domain attached to diverse structurally distinct tail domains. Newly found myosins, called unconventional myosins, are different from conventional thick filament forming myosin and play a fundamental role in various types of cell motility/contractility. therefore, malfunction or mutation of these motor proteins would cause several health problems. Among unconventional myosins, class I myosin is widely distributed among tissues and thought to play a important role on chemotaxi, phagocytosis, secretion, endocytosis, vesicular trafficking, mechanochemical regulation of Ca2+ channels in hair cells, etc. The major flaw of unconventional myosin research is the lack of knowledge about the molecular and cellular function and regulation of these molecules. The proposed project studies the molecular mechanism of regulation of mammalian myosin Is and the cellular function of myosin Is. Of interest is the fact that many unconventional myosins contain multiple calmodulins as light chain subunits. This raises the hypothesis that Ca2+ binding to calmodulin plays a critical role in the regulation of motor function. The project will investigate the mechanism by which myosin I activity is regulated by Ca2+. It has become clear that several different isoforms of myosin I are present within the same cell type and it is hypothesized that each isotype plays a distinct physiological role in the motile processes of cells. The project will address this question by: determining the motor characteristics of each isoform, which should be closely related to the requirements of a particular cellular motile process; identifying the distinct intracellular localization and the translocation/movements of myosin Is during motile/contractile processes; and identifying the specific targeting proteins of myosin Is. To accomplish these goals, recombinant DNA technology and 3D digital imaging techniques will be employed along with various protein biochemical, biophysical and cell biological techniques. The analysis of the motor activity will be done with 1) invitro actin sliding motility assay; 2) a feedback enhanced laser trap system as a measure of force production. The intracellular localization and movement of myosin I will be done with 3D fluorescence digital imaging techniques and the myosin I targeting proteins will be identified using the yeast two hybride system and protein biochemical means.
