Mechanisms of growth cone turning in diffusible gradient

描述（由申请人提供）：细胞感知环境和确定细胞外刺激的方向和接近度的能力，以及随之而来的正确运动，不仅对神经发育（例如神经元迁移和生长锥引导）至关重要，而且对免疫、血管生成、伤口愈合和胚胎发生也至关重要。细胞定向运动对许多病理事件，特别是癌细胞转移也至关重要。因此，更好地理解细胞对细胞外刺激的定向反应背后的细胞机制，将构成我们对定向细胞运动的基本知识的重大进步，并可以为许多疾病的发展策略和治疗提供基础。本研究将以神经生长锥为模型，研究细胞在响应细胞外信号时定向运动的时空信号转导和细胞骨架机制。最近的研究发现，肌动蛋白解聚因子(ADF)/cofilin家族蛋白是控制细胞定向运动的肌动蛋白动力学的关键调节因子。我们最近的数据表明，LIM激酶（LIMK）和弹弓磷酸酶（SSH）的平衡作用控制了局部磷酸化依赖的ADF/cofilin活性，从而引发生长锥的双向转动。此外，已发现ADF/cofilin及其上游效应物LIMK1是局部合成的，其局部翻译可由microrna调控。因此，我们认为ADF/cofilin是生长锥寻径过程中复杂信号网络的会聚目标，局部ADF/cofilin活性控制肌动蛋白动力学和生长锥转向，以响应细胞外引导信号。此外，ADF/cofilin及其上游调节因子LIMK1的局部翻译可以显著影响ADF/cofilin对肌动蛋白动力学的细胞功能，并有助于生长锥的定向运动。本应用旨在测试ADF/cofilin的作用及其通过磷酸化和局部翻译调控生长锥对细胞外信号的定向反应。利用良好定义的神经元培养系统进行生长锥转向试验，复杂的高分辨率成像技术，直接操纵细胞内信号，以及信号级联的分子和药理学操作，我们将解决四个具体目标：(1) ADF/cofilin在生长锥定向导向中的精确作用；(2)LIM激酶1 （LIMK1）和Slingshot磷酸酶（SSH）在ADF/cofilin调控和生长锥转向中的作用；(3)ADF/cofilin和LIMK1局部合成对生长锥导向的贡献；(4)ADF/cofilin及其磷酸化和局部合成调控在视网膜轴突寻路中的体内作用。我们的目标是了解ADF/cofilin家族蛋白的时空调节如何将细胞外信号转化为生长锥的定向运动，这将为许多生理和病理事件中定向细胞运动的机制提供重要的见解。

DESCRIPTION (provided by applicant): 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.