Directed growth cone migration by calcium signals

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定向运动的时空钙信号机制。钙是一种关键的第二信使，调节各种细胞运动，包括定向细胞迁移。它已被确定，Ca 2+介导的生长锥响应的指导线索，包括吸引和排斥的转向反应。最近的研究表明，不同的，本地化的Ca 2+信号引起的钙-钙调蛋白依赖性激酶II（CaMKII）和钙调磷酸酶（CaN）的活性的平衡作用，以控制的吸引和排斥旋转的生长锥。本申请旨在进一步评估控制双向生长锥转向响应于引导线索的Ca 2+机制。本研究的主要目的是：（1）研究细胞内Ca ~（2+）信号的时空模式及其在控制生长锥转向中的作用;（2）研究感受不同Ca ~（2+）信号以控制生长锥转向的下游机制;（3）验证FAK/Src在生长锥导向中将Ca ~（2+）信号与酪氨酸磷酸化联系起来的假说。拟议的研究将利用我们严格的生长锥转动试验和高分辨率数字成像，笼状化合物的光活化和信号成分的分子操作的组合。特别是，直接操纵细胞内的Ca 2+浓度的聚焦激光诱导的光解（FLIP）的笼状Ca 2+将被广泛用于解剖的信号组件。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Ca 2+信号转导机制。长期目标是了解允许轴突生长锥通过复杂的细胞外空间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推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认识，而且还将为重要的生物反应过程中迁移细胞定向传感的细胞机制提供重要的见解，例如炎症反应过程中白细胞的趋化性。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定向运动的时空钙信号机制。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见解。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 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.