The neuronal growth cone performs several key functions in the establishment, remodeling and repair of the nervous system. The growth cone is involved in elongation of the neurite, changing direction of its advance, recognizing cues from the molecular terrain, and in synaptogenesis. The present proposal will test the idea that critical aspects of the growth cone's performance rely upon partitioning duties to different sites; in particular, this work focuses on the fan-like filopodia that lead the advancing growth cone into new cellular environments. Recent high resolution microscopic observations have demonstrated that activities associated with even a single filopodium can substantially alter events within the growth cone proper. Such findings suggest a critical role for filopodia in several key developmental processes. This proposal tests a series of interrelated hypotheses which taken together suggest that filopodia serve multiple, quite different roles in neural development. We will investigate: The long standing proposal that a major role for filopodia is as antennae-like sensors of the environment. We will then investigate how the growth cone integrates filopodial inputs in order to distinguish between those environmental cues that produce minor course corrections in navigation (i.e., guidance) versus complete inhibition of growth cone motility (i.e., collapse). A rigorous pharmacological dissection will then defend the intracellular pathways underlying the amplification of filopodial calcium second messenger signals. These three investigations will provide insights into how information can be transduced from the environment into meaningful signals within the neuronal growth cone. Finally, we will investigate a process with the high resolution afforded by cell culture which has previously only been studied in situ and is based solely on histological evidence. That is, does an alternative mechanism of target localization exist whereby backbranches can be formed along the neurite? Such backbranches could then result in the generation of new neurites that reach the inappropriate target. This latter set of experiments investigates an important addition to the conventional view that the activities of the lead growth cone are the primary determinants of target localization. Taken together, the proposed research directly tests a series of related hypotheses that will elucidate the multiple roles served by filopodia at key times in neural development and regeneration. By employing newly developed preparations that allow high resolution analyses of several different functions and focussing these around fundamental issues of early development, these experiments will yield an integrated picture of the multiple contributions that filopodia make to the overall function of neuronal growth cones.
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