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: some of these receptors interact with extracellular matrix components that may serve as spatial cues, still others recognize cell adhesion molecules (CAMs) on other cell surfaces. 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 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 molecular dynamics underlying the motility process itself. The results of this work have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and will extend our understanding of the process of nerve regeneration. Diagnostic probes for developmental and regenerative neuronal disorders could also result from the proposed research. This project relies on the use of high spatial and temporal resolution digital imaging techniques to investigate the behavior of membrane proteins involved in growth cone guidance and synaptogenesis. To achieve these ends, a system utilizing pseudosubstrate probes has been developed that allows tracking of membrane proteins in growth cones. These probes (typically 100-200 nm beads derivatized with ligands of interest) are being used to investigate alterations of growth cone cytoskeletal structure and motility that occur both in response to the pseudosubstrates themselves and during growth cone target interactions. To facilitate the pseudosubstrate experiments, a single beam gradient optical trap (laser tweezers) will be constructed. The laser tweezers is a non invasive method for micropositioning of small objects (like microbeads) which can also be used to measure forces associated with the growth cone guidance and recognition processes. To compliment these studies, intracellular actin dynamics will be characterized using fluorescence photoactivation techniques. This will allow us to compare and contrast intracellular f-actin and cell surface protein movements involved in growth cone target recognition or substrate adhesion. Finally, a reverse genetic approach will be used to generate specific molecular probes for proteins likely to be involved in regulation of growth cone motility. Specifically, fusion proteins immunogens expressed subsequent to gene cloning from an Aplysia cDNA library will be used to generate antibodies to CAM and actin binding proteins of interest.
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