Understanding how and why the growth cone moves will shed light on the fundamental processes of neurodevelopment and may also suggest strategies to induce growth cone motility and regeneration in damaged neurons. While much progress has been made in identifying growth cone guidance signals and their receptors, relatively little is known about how these signals are transduced into the changes in cytoskeletal dynamics that are required for directed growth cone motility. It is clear, however, that multiple guidance signals converge on the cytoskeleton and must ultimately be integrated into a coordinated response. Proteins that bind to and directly regulate the cytoskeleton may thus serve as the ultimate interpreters of guidance signals. The overall goal of this proposal is to identify key growth cone cytoskeletal regulatory proteins and determine their role in growth cone motility and guidance. Experiments will focus on the Arp2/3 complex, which nucleates the formation of branched actin filaments and plays an essential role in many types of actin-based cell motility. Recent findings indicate that Arp2/3 is a negative regulator of growth cone translocation and that Arp2/3-dependent actin structures play an important role in coordinating actin and microtubule dynamics in the growth cone. We will test the hypothesis that Arp2/3 regulates cytoskeletal dynamics in response to guidance signals. A combination of molecular and biochemical techniques, live cell imaging and correlative electron microscopy will be used to deduce the mechanism of Arp2/3 function in growth cone motility and pathfinding. Tissue specific inhibition of Arp2/3 will be used to characterize the role of Arp2/3 in the developing nervous system. Finally, identification of the proteins that activate Arp2/3 in growth cones will provide insight into how Arp2/3 is regulated by upstream signaling pathways.
