Analysis of myosin II function in neuronal growth cones

Growth cone motility and guidance must ultimately be understood in terms of effects upon dynamic actin and microtubule cytoskeletons. Myosin II is a mechanoenzyme important for exerting traction forces upon the actin cytoskeleton that underlies cell motility. This dissertation investigates myosin II's role in organizing a dynamic actin cytoskeleton within Aplysia growth cones. A neuronal myosin II homolog was cloned from Aplysia and specific antisera were developed for motility studies.; Retrograde actin flow works in concert with cell adhesion to generate traction forces involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report myosin II inhibition alone decreases retrograde flow by 51% and remaining flow can be almost fully accounted for by the "push" of plus end actin assembly at the leading edge. Interestingly, actin bundles associated with filopodium roots elongated by ∼83% after myosin II inhibition. This unexpected result was due to decreased rates of actin bundle severing near their proximal (minus/pointed) ends which reside in the growth cone transition zone. These studies reveal a novel mechanism for regulation of actin bundle length by myosin II dependent actin bundle severing and demonstrate retrograde flow is a steady state that depends on both myosin II contractility and actin network treadmilling.; Aplysia growth cones respond to permissive extracellular target substrates with coordinated changes in cytoskeletal dynamics and motility that mediate target site directed advance. Acto-myosin adhesion complexes form at target sites during growth cone advance responses using a restrained bead interaction (RBI) assay. Inhibition of ROCK potentiated growth cone advance by decreasing response latency times. After complete inhibition of myosin II with blebbistatin some microtubule targeting was observed but interactions never completed. In addition, active Src kinases localize normally to adhesion scaffolds around restrained beads, but further development of adhesion sites require myosin II.