Regulation of Neuronal Motility

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DESCRIPTION (provided by applicant): Understanding the molecular events that underlie the process of growth cone guidance and axonal pathfinding in the developing brain are one of the major challenges for Neurobiology. Recently, research in developmental neurobiology has moved decisively into the molecular arena with characterization of molecules that act as extracellular pathfinding cues and specific receptors on growth cones and axons for recognizing these cues. Some of these receptors interact with extracellular components that may serve as spatial cues, others recognize cell adhesion molecules (CAMs) on other cells and still other receptors respond to diffusable chemotropic signals. Neuronal growth cones mediate axon guidance by sensing and responding "intelligently" to these diverse biochemical cues with appropriate changes in their motility and structure. Such effects ultimately involve translation of signals received at the membrane into appropriate changes in cytoskeletal protein dynamics that underlie the growth cone's motility response. In the last decade, there has been intense interest and rapid progress made characterizing the cell surface molecules and ligands involved in growth cone guidance. In addition, the complex web of signal transduction pathways that transmit these signals to downstream effectors has begun to emerge. With all this progress, however, our understanding of how the cytoskeletal machinery is affected by these pathways is still quite limited. This has been in great part due to lack of appropriate bioassays for measuring cytoskeletal protein function in living cells. Recent advances in fluorescent molecular probes and digital imaging have overcome critical limitations in this area. In particular, it is now possible to measure rates of assembly, disassembly and translocation of cytoskeletal proteins in a living cell and correlate cytoskeletal dynamics will the structural changes involved in various forms of motility. Mounting evidence suggests the importance of bi-directional cross talk between dynamic actin and microtubule (MT) cytoskeletal networks in directed cell movements including growth cone guidance. In the neuronal growth cone, a sharp, yet highly dynamic, interface exists between elongating axonal microtubules and peripheral actin networks that support exploratory behavior and provide signals involved in guiding axonal advance. The work proposed here addresses how MTs and actin filaments interact with one another in real time in living growth cones and the functional significance of such interactions. This investigation will provide information fundamental to our understanding the cell biology of growth cone motility since simultaneous analysis of actin filament and MT dynamics has never been achieved a growth cone to date. To reach this goal, multimode Fluorescent Speckle Microscopy (FSM) will be used. Using this technology a full quantitative characterization of how actin filament dynamics affect microtubule behavior, and conversely, how microtubule dynamics affect actin filament behavior will be done. With this knowledge in hand, the more complex problem of how MT and actin filament systems interact and affect one another during growth cone guidance events will be tackled. This will include evaluation of how traction forces that develop in peripheral actin networks bias MT advance, and if tension affects MT polymer dynamics. We will also investigate how protein kinase signaling pathways important in regulation of axon guidance modulate behavior of the cytoskeletal effector machinery during target interaction events. Information gleaned from these studies should have a fundamental impact on our understanding of the cell biology of axon guidance and nerve regeneration.