Mechanisms of growth cone turning in diffusible gradient

描述（由申请人提供）：细胞感知环境并确定细胞外刺激的方向和接近度以及随后的正确运动的能力不仅对于神经发育（例如神经元迁移和生长锥引导）而且对于免疫、血管生成、伤口愈合和胚胎发生都是基本的。定向细胞运动对于许多病理事件也是至关重要的，特别是癌细胞转移。因此，更好地理解细胞对细胞外刺激的定向反应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，研究细胞外信号诱导下神经生长锥定向运动的时空信号转导和细胞骨架机制。最近的研究已经确定了肌动蛋白解聚因子（ADF）/cofilin蛋白家族作为控制定向细胞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ADF/cofilin的局部磷酸化依赖性活性，以引起生长锥的双向转动。此外，ADF/cofilin及其上游效应子LIMK 1也被发现是局部合成的，其局部翻译受microRNA的调控。因此，我们建议，ADF/cofilin是一个复杂的信号网络的收敛目标，在生长锥寻路和本地ADF/cofilin活动控制肌动蛋白动力学和生长锥转向响应细胞外的指导线索。此外，ADF/cofilin及其上游调控因子LIMK 1的局部翻译可显著影响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。本申请旨在测试ADF/cofilin的作用及其通过磷酸化和局部翻译在生长锥对细胞外信号的定向反应中的调节。利用生长锥转动试验的明确神经元培养系统、复杂的高分辨率成像技术、细胞内信号的直接操纵以及信号级联的分子和药理学操纵，我们将解决四个具体目标：（1）ADF/cofilin在生长锥方向操纵中的精确作用，（2）LIM激酶1（LIMK 1）和弹弓磷酸酶（SSH）在ADF/cofilin调节和生长锥转向中的作用，（3）ADF/cofilin和LIMK 1的局部合成对生长锥转向的贡献，（4）ADF/cofilin在视网膜轴突寻路中的作用及其磷酸化和局部合成的调节。我们的目标是了解ADF/cofilin蛋白家族的时空调控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。 公共卫生相关性：细胞感知环境并确定细胞外刺激的方向和接近度以及随后的正确运动的能力不仅对于神经发育（例如神经元迁移和生长锥引导）而且对于免疫、血管生成、伤口愈合和胚胎发生都是基本的。定向细胞运动对于许多病理事件也是至关重要的，特别是癌细胞转移。因此，更好地理解细胞对细胞外刺激的定向反应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，研究细胞外信号诱导下神经生长锥定向运动的时空信号转导和细胞骨架机制。这组研究的结果将为生长锥寻路的细胞机制提供重要的见解，同时也指导了许多生理和病理事件中的细胞运动。因此，这项工作直接关系到公众健康。

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.