The experiments described in this proposal are designed to investigate the roles of actin-based motor proteins - myosins - in filopodial retraction and extension, neurite outgrowth and neuronal guidance using cultured chick dorsal root ganglion (DRG) neurons in combination with the unique technique, CALl (chromophore-assisted laser inactivation). First, the ability of CALI to inhibit myosin I, II and V's actin stimulated MgATPase activity and actin-dependent motility will be examined in vitro using malachite green-labeled anti-myosin antibodies. Second, the growth cones of DRG neurons loaded with malachite green-labeled anti-myosin antibodies will be completely or partially laser irradiated and the effects of laser irradiation on growth cone motilities will be recorded. The rates of the filopodial extension and retraction in the laser irradiated areas of the growth cones will be calculated and compared with that of non-irradiated areas as well as neurons loaded with malachite green-labeled non-specific IgG. Third, the polystyrene microbead assay will be employed to examine the involvement of myosins in the retrograde movement of F-actin in single filopodia. Four, the neurons will be loaded with malachite green-labeled anti-myosin antibodies in various combinations and inactivate the activities of two or three myosin forms together. The effects of inactivation of more than one myosin forms will be determined and compared with the results collected from the previous approaches. The proposed work will be the first demonstration of the role of myosins in growth cone motility. The elucidation of the functions of Myosins will enhance our understanding of the molecular mechanisms underlying axon growth and the neuronal guidance.
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