): The unconventional myosin, myosin-VIIa is critical for the development and function of the inner ear and for the maintenance and function of the retina. Mutations in myosin-VIIa result in Usher disease, characterized by profound deafness and retinal degeneration. Myosin-VIIa mutations also cause recessive and dominant deafness in humans and deafness in the mouse. Classically, myosins are described as motors that hydrolyze ATP and transport protein or organelle cargo along actin filaments. Various genetic and localization studies hypothesize that myosin-VIIa may act in protein transport, organelle movement, phagocytosis, endocytosis, or assembly of the inner ear actin cytoskeleton. Although this list is impressive, we don't actually know if myosin-VIIa is a motor and, significantly, no protein or organelle cargo have been identified. Our long term goal is to elucidate the function of myosin-VIIa in sensory cells and to identify the molecular cargo that this motor is transporting. Towards this aim, we will purify myosin-VIIa and investigate its mechanochemical properties using in vitro actin-binding, ATPase, and motility assays. We will use mutagenesis studies to characterize the effects of Usher disease mutations on myosin-VIIa enzyme function in vitro. To determine the molecular cargo of myosin-VIIa, we will continue our analysis of three Myosin interacting Proteins, MYP6, MYP13 and MYP-kelch, which we identified as proteins that bind to the tail of myosin-VIIa. We will determine how these MYPs assemble with myosin-VIIa in vivo and also assess their roles in myosin-VIIa function. Ultimately we will ascertain whether binding of molecular cargo directly affects myosin motility. To investigate the functions of myosin-VIIa, we have identified two retina pigmented epithelium cell lines that recreate the expression of myosin-VIIa seen in vivo. Using transfection methods we will experimentally assess the hypotheses that myosin-VIIa functions in cytoskeletal assembly, phagocytosis, and membrane transport. Finally, we will extend upon our observation that myosin-VIIa is phosphorylated in vivo, by investigating the importance of this phosphorylation on myosin-VIIa function in the cultured retina lines.
