Understanding how axons respond to guidance cues to navigate through the developing embryo to connect with the proper target is essential to combat developmental nervous system disorders and regeneration. Sema3A is a common axon repellent which causes growth cone collapse and axon paralysis in numerous types of neurons. To date, not much is known about the intracellular mechanisms that cause cytoskeletal rearrangements necessary for collapse. The collapsing-response-mediator-protein (CRMP) family of cytosolic phosphoproteins is required for Sema3A-induced growth cone collapse. This proposal aims to determine the mechanism of CRMP function at molecular, biochemical, and functional levels of analysis. First, the solved crystal of CRMP1 will be used to generate a series of targeted mutant proteins. These proteins will be used to map functional domains on CRMP1 surface. Second, a constitutively active (CA) mutant CRMP1 that we developed will be used to identify novel CRMP1-binding proteins. Interactions between CRMP1 and putative interacting proteins will be characterized and relevance to Sema3A signaling will be determined. Finally, gain-of-function (via overexpression of CA CRMP1) and loss-of function (RNAi) experiments will be performed to assess the function of CRMP proteins in intact chick embryos. The proposed plan of research will elucidate the mechanism of CRMP action in Sema3A signal transduction at molecular, biochemical, and functional levels of analysis.
