During the pathfinding phase of neuronal development, the growth cone functions as a specialized sensor, capable of guiding extending axons toward distant target sites. Growth cones display a high level of actin-based motility which, in an as yet unspecified manner, supports this guidance process. Specific receptors on the growth cone surface also appear to be involved in growth cone guidance. Some of these receptors interact with components of the extracellular matrix that may serve as spatial cues. Although there has been intense interest in characterizing molecules involved in neuronal guidance, very little is known about the actual signal transduction processes involved; for example, how receptor occupation leads to dynamic alterations of cytoskeletal structure and motility likely to underlie pathfinding decisions. The proposed research attempts to fill this gap in our knowledge: (1) by characterizing signal transduction mechanisms involved in regulation of growth cone motility and structure and (2) by investigating the basic mechanochemical processes that sustain this motility. The results of this work should have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and may also exceed our understanding of the process of nerve regeneration. Diagnostic probes for molecular substrates involved in developmental and regenerative neuronal disorders could also result from the proposed research. A model system employing high resolution digital-video imaging of microscopic membrane surface probes in living neurons will be used to investigate signal transduction mechanisms involved in growth cone guidance. Specific probes for receptors thought to be involved in the processes of guidance and target recognition will be developed. These membrane probes will then be used to investigate alterations of growth cone cytoskeletal structure and motility that appear to occur in response to appropriate extracellular guidance cues. The ultimate goal of the signal transduction section of this project is to characterize in detail receptor mediated processes that regulate membrane-cytoskeletal interactions and actomyosin-based motility in growth cones and relate these molecular events to the macroscopic problem of growth cone guidance. How growth cones generate locomotive force is not known; thus, the second phase of this proposal involves characterization of the mechanochemical processes and cytoskeletal dynamics underlying growth cone motility. A clearer understanding of growth cone mechanochemistry will compliment the signal transduction questions outlined above. The identity, spatial localization and biochemical properties of neuronal myosin-like molecules involved in actin-based movements in growth cones will be investigated. Neuronal actin-myosin interactions will be approached by attempting to reconstitute neuronal actin-myosin motility in vitro in a demembranated cell model system. Further characterization of actin dynamics in intact growth cones will also be undertaken using fluorescent analog cytochemistry.
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