The cell's ability to sense the environment and to determine the direction and proximity of an extracellular stimulus, followed by correct movement, is fundamental not only for neural development (e.g. neuronal migration and growth cone guidance) but also for immunity, angiogenesis, wound healing, and embryogenesis. Directional cell movement is also crucial for many pathological events, especially cancer-cell metastasis. Therefore, a better understanding of the cellular mechanisms that underlie the directional responses of cells to extracellular stimuli would constitute a major advance of our basic knowledge on directional cell motility and could provide the foundation for developing strategies and treatments for many illnesses. The proposed study will use nerve growth cones as the model to study the spatiotemporal signal transduction and cytoskeletal mechanisms underlying directional motility in response to extracellular cues. Recent studies have identified the actin depolymerizing factor (ADF)/cofilin family of proteins as the key regulator of the actin dynamics that controls directional cell motility. Our recent data indicate that a balancing act of LIM kinase (LIMK) and slingshot phosphatase (SSH) controls the local phosphorylation-dependent activity of ADF/cofilin to elicit bidirectional turning of the growth cone. In addition, ADF/cofilin and its upstream effector LIMK1 have been found to be locally synthesized and the local translation can regulated by microRNAs. We therefore propose that ADF/cofilin is a converging target of intricate signaling networks during growth cone pathfinding and local ADF/cofilin activity controls the actin dynamics and growth cone steering in response to extracellular guidance cues. Furthermore, local translation of ADF/cofilin and its upstream regulator LIMK1 could significantly impact the cellular functions of ADF/cofilin on the actin dynamics and contribute to the directional motility of growth cones. This application is to test the roles of ADF/cofilin and its regulation by phosphorylation and local translation in directional responses of growth cones to extracellular cues. Taking advantage of a well- defined neuronal culture system for growth cone turning assays, sophisticated high- resolution imaging techniques, direct manipulation of intracellular signals, and molecular and pharmacological manipulation of signaling cascades, we will address four specific aims: (1) the precise role of ADF/cofilin in growth cone directional steering, (2) the role of LIM kinase 1 (LIMK1) and Slingshot phosphatase (SSH) in ADF/cofilin regulation and growth cone turning, (3) the contribution of local synthesis of ADF/cofilin and LIMK1 to growth cone steering, and (4) the in vivo roles of ADF/cofilin and its regulation by phosphorylation and local synthesis in retinal axon pathfinding. Our goal is to understand how spatiotemporal regulation of ADF/cofilin family of proteins translates extracellular cues to directional movement of the growth cone, which would provide significant insights into the mechanisms of directed cell movement in many physiological and pathological events. ? Public Health Relevance: The cell's ability to sense the environment and to determine the direction and proximity of an extracellular stimulus, followed by correct movement, is fundamental not only for neural development (e.g. neuronal migration and growth cone guidance) but also for immunity, angiogenesis, wound healing, and embryogenesis. Directional cell movement is also crucial for many pathological events, especially cancer-cell metastasis. Therefore, a better understanding of the cellular mechanisms that underlie the directional responses of cells to extracellular stimuli would constitute a major advance of our basic knowledge on directional cell motility and could provide the foundation for developing strategies and treatments for many illnesses. The proposed study will use nerve growth cones as the model to study the spatiotemporal signal transduction and cytoskeletal mechanisms underlying directional motility in response to extracellular cues. The results from this set of studies will provide significant insights into the cellular mechanisms of growth cone pathfinding, but also directed cell movement in many physiological and pathological events. Therefore the work is directly relevant to public health. ? ?
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