Mechanisms of growth cone turning in diffusible gradient

项目摘要 细胞感知环境和确定方向的能力以及 接近细胞外刺激，然后是正确的运动，是根本不是 仅适用于神经发育(例如神经元迁移和生长锥引导)，但 也用于免疫、血管生成、伤口愈合和胚胎发育。定向单元 运动在许多病理事件中也是至关重要的，尤其是癌细胞转移。 因此，对细胞机制的更好理解 细胞对细胞外刺激的方向性反应将构成 我们对定向细胞运动的基本知识，并可以为 开发多种疾病的策略和治疗方法。拟议的研究将使用 以神经生长锥体为模型研究神经的时空信号转导 响应细胞外暗示的定向运动潜在的细胞骨架机制。 最近的研究发现肌动蛋白解聚因子(ADF)/cofilin家族 作为控制细胞定向运动的肌动蛋白动力学的关键调节器的蛋白质。 我们最近的数据表明，LIM激酶(LIMK)和弹弓的平衡行为 磷酸酶(SSH)控制ADF/cofilin的局部磷酸化依赖活性 以引发生长锥体的双向旋转。此外，ADF/cofilin及其上游 已经发现效应器LIMK1是局部合成的，并且局部翻译可以 受microRNAs调控。因此，我们认为ADF/cofilin是一个融合的靶点。 在生长锥寻路和局部ADF/Cofilin过程中的复杂信号网络 细胞外的活性控制着肌动蛋白的动态和生长锥体的调控 引导线索。此外，ADF/cofilin及其上游调控因子的本地翻译 LIMK1可显著影响ADF/cofilin对肌动蛋白的细胞功能 并有助于生长球果的定向运动。此应用程序将用于 检测ADF/cofilin的作用及其通过磷酸化和局部翻译的调节 生长锥对细胞外暗示的方向性反应。利用一口井- 用于生长锥体翻转分析的定义神经元培养系统，复杂的高密度 分辨率成像技术、细胞内信号的直接操作和分子 和药物操纵信号级联，我们将解决四个具体的 目的：(1)ADF/Cofilin在生长锥定向导向中的精确作用；(2)ADF/Cofilin在生长锥定向引导中的作用 LIM激酶1(LIMK1)和Slingshot磷酸酶(SSH)在ADF/cofilin调节和 生长锥体转动，(3)ADF/cofilin和LIMK1的局部合成对细胞生长的贡献 以及(4)ADF/cofilin在体内的作用及其调节。 视网膜轴突通路中的磷酸化和局部合成。我们的目标是 理解ADF/cofilin蛋白家族的时空调控如何翻译 生长锥体定向移动的细胞外信号，这将提供 在许多生理学研究中对定向细胞运动机制的重要见解 以及病态事件。

Project Summary 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.