The long-range goal of this research is to better understand how signal transduction cascades lead to cytoskeletal rearrangements that are necessary for guided axon outgrowth. Guidance of growth cones to their targets occurs through specific interactions of extracellular ligands with receptors on the surface of growth cones. While large families of diffusible, cell surface and extracellular matrix (ECM) bound ligands and their receptors have been identified, less is known of the intracellular signaling cascades and cytoskeletal rearrangements that occur downstream of receptor-ligand interactions. In particular, very little is understood about how growth cones integrate signals generated through interactions with multiple ligands, which likely occur in dynamic combinations of molecular gradients, guideposts and boundaries in the developing nervous system. Interactions among intracellular signaling intermediaries such as calcium, cAMP, protein tyrosine kinases and Rho family GTPases are likely to play important roles in regulating pathfinding decisions. These signals in turn organize cytoskeletal elements that regulate actin/microtubule polymerization, membrane protrusion, focal complex formation, substrata adhesion, and de-adhesion, which are all essential processes for proper control over growth cone motility. Our recent work identified Src family kinases as important mediators of axon outgrowth, which are negatively regulated by calcium-activated calpain. In this application we propose to further investigate the role of Src kinases as key intermediaries between axon guidance cues and effectors of cytoskeletal dynamics, which include Rho GTPases and their downstream targets. Specifically, we propose to: 1) Examine in live growth cones how chemotropic axon guidance cues and classic second messengers modulate Src-dependent tyrosine phosphorylation. 2) Test the functional regulation of N-WASP and mDia2 by Src kinases and Cdc42 in growth cones. 3) Determine the role of phosphotyrosine-containing point contacts in the control of growth cone turning in vitro. A better understanding of signal transduction in growth cones will provide insight into the molecular basis of developmental and neurological disorders.
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