Directed growth cone migration by calcium signals

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。该研究将使用神经生长锥作为模型来研究响应细胞外信号的定向运动的时空Ca2+信号机制。钙是调节多种细胞运动的关键第二信使，包括定向细胞迁移。已经确定Ca2+介导生长锥对引导信号的反应，包括吸引和排斥的转向反应。最近的研究表明，不同的局部Ca2+信号引起钙调蛋白依赖性激酶II （CaMKII）和钙调磷酸酶（CaN）磷酸酶活性的平衡作用，以控制生长锥的吸引和排斥转向。本应用旨在进一步评估Ca2+机制，控制双向生长锥转向响应的指导线索。提出了三个具体目标：(1)研究胞质Ca2+信号的时空模式及其在控制生长锥转向中的作用；(2)研究感知各种Ca2+信号以控制生长锥转向的下游机制；(3)验证FAK/Src将Ca2+信号与生长锥引导中酪氨酸磷酸化联系起来的假设。拟议的研究将利用我们对生长锥转动的严格分析，以及高分辨率数字成像、笼化化合物的光激活和信号成分的分子操作的结合。特别是，通过聚焦激光诱导的Ca2+光解（FLIP）直接操纵细胞内Ca2+浓度将被广泛用于剖析信号传导成分。总之，这些实验代表了一项全面的研究，旨在了解生长锥运动和指导的Ca2+信号传导机制。长期目标是了解允许轴突生长锥在复杂的细胞外空间中导航以建立复杂连接的分子和细胞机制。这项研究的结果不仅将促进我们对大脑发育和恢复过程中精确神经元布线的分子机制的了解，而且还将为在重要的生物反应中迁移细胞定向传感的细胞机制提供重要的见解，例如炎症反应中白细胞的趋化性。公共卫生相关性：细胞感知环境和确定细胞外刺激的方向和接近程度的能力，以及随之而来的正确运动，不仅对神经发育（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。该研究将使用神经生长锥作为模型来研究响应细胞外信号的定向运动的时空Ca2+信号机制。这组研究的结果将为生长锥寻路的细胞机制以及许多生理和病理事件中的定向细胞运动提供重要的见解。因此，这项工作与公共卫生直接相关。

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 Ca2+ signaling mechanisms underlying directional motility in response to extracellular cues. Calcium is a key second messenger that regulates a variety of cell motility, including directed cell migration. It has been established that Ca2+ mediates growth cone responses to guidance cues, including attractive and repulsive turning responses. Recent studies indicate that different, localized Ca2+ signals elicit a balancing act on the activity of calcium-calmodulin- dependent kinase II (CaMKII) and Calcineurin (CaN) phosphatase to control the attractive and repulsive turning of the growth cone. This application aims to further evaluate the Ca2+ mechanisms that control bidirectional growth cone steering in response to guidance cues. Three specific aims are proposed: (1) to examine the spatiotemporal patterns of cytosolic Ca2+ signals and their role in controlling growth cone steering, (2) to investigate the downstream mechanisms that sense various Ca2+ signals to control growth cone turning, (3) to test the hypothesis that FAK/Src links Ca2+ signaling to tyrosine phosphorylation in growth cone guidance. The proposed studies will take advantage of our rigorous assays of growth cone turning and a combination of high-resolution digital imaging, photoactivation of caged compounds, and molecular manipulation of signaling components. In particular, direct manipulation of intracellular Ca2+ concentrations by focal laser-induced photolysis (FLIP) of caged Ca2+ will be extensively used for dissecting the signaling components. Together, these experiments represent a comprehensive study that aims to understand the Ca2+ signaling mechanisms underlying growth cone motility and guidance. The long-term goal is to understand the molecular and cellular mechanisms that allow axonal growth cones to navigate through complex extracellular spaces for establishing intricate connections. Results from this study will not only advance our knowledge of molecular mechanisms underlying precise neuronal wiring during brain development and recovery, but also provide important insights into the cellular mechanisms underlying directional sensing of migrating cells during important biological responses such as chemotaxis of leukocytes during inflammatory response. 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 Ca2+ signaling 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, as well as of directed cell movement in many physiological and pathological events. Therefore the work is directly relevant to public health.