Coordination between Actin Polymerization and Network Contractility Regulates Growth Cone Motility

Proper neurite guidance is important for wiring the nervous system during development and regeneration. Growth cones are motile sensory structures at the tips of neurites responsible for receiving and interpreting guidance cues. Growth cones rely on coordinated regulation of actin filament dynamics to maintain structural integrity and coherent motility. Under control conditions, the growth cone actin cytoskeleton exists in a steady state with continuous polymerization in the front and filament recycling more proximally. Filopodial bundles and veil networks assembled at the leading edge are transported rearward via retrograde flow. Myosin II contractility drives flow and condenses the network into contractile structures referred to as actin arcs at the transition (T) zone between the peripheral (P) and central (C) domains. Arcs move centripetally into the contractile node at the growth cone neck. As filament translocation and reorganization link the actin network into an interconnected entity, significant interactions must exist among network polymerization, turnover, and contractility. Even though the biochemical properties of individual actin regulators have been extensively characterized, how they functionally interact on the cellular scale is not well understood.;The first part of this thesis examines how Arp2/3 complex dependent actin polymerization and myosin II contractility coordinate to maintain veil structure and dynamic behavior. Arp2/3 complex, a key actin nucleator in motile cells, is crucial for the formation of veil actin networks. Kinetic signatures of Arp2/3 complex inhibition depend on myosin II. At normal levels of myosin II activity, P-domain retrograde flow increases and veils retract with Arp2/3 complex inhibition. The effects are reversed when myosin II activity is low. These results point to a novel function of actin networks assembled by Arp2/3 complex as restraining myosin II contractility, which may be modulated to promote protrusion or resist retraction.;The second part examines how a transducer of repulsive guidance signals, protein kinase C (PKC) affects both Arp2/3 complex dependent actin polymerization and myosin II driven contractility to induce neurite retraction. PKC's two-pronged mechanism of action is spatially separated but functionally synergistic. In the P-domain, PKC activation depletes Arp2/3 complex and barbed end actin nucleation sites, reducing the density of actin veil networks. In the C-domain, PKC increases myosin II regulatory light chain phosphorylation through CPI-17. Combination of decreased network resistance and increased myosin II activity produces a hyper-contractile state, impairing neurite outgrowth. These results provide a mechanistic framework for understanding PKC action in growth cone motility, which may be exploited in therapeutic interventions for neuronal regeneration.