Mechanisms of Growth Cone Turning in Diffusible Gradient

描述（由申请人提供）：特定神经元连接的形成需要通过各种表面结合和扩散的引导线索准确地引导轴突发育到正确的目标。轴突引导对于脑损伤和疾病后受损神经元连接的功能修复也至关重要。在引导过程中，轴突尖端的活动生长锥从细胞外空间感知方向，并引导轴突通过发育胚胎的复杂环境到达其特定目标。近年来，各种表面结合和扩散引导分子的分子鉴定取得了重大进展。然而，细胞机制的方向传感和导向的生长锥仍然很大程度上未知。提出的研究旨在了解生长锥如何响应不同类型的可扩散引导分子，以引导到特定方向。需要验证的工作假设如下：不同的细胞外信号可以通过不同的信号传导途径起作用，但它们都引发了局部的信号级联反应，这些信号级联反应以空间和时间限制的方式靶向细胞骨架，从而导致生长锥向特定方向移动。利用爪蟾生长锥的大尺寸、生长锥转向的体外严格检测系统、高分辨率数字成像和细胞内操作技术的结合，研究小组计划彻底研究与生长锥的特定转向行为（吸引或排斥）相关的细胞成分。拟议的项目有四个具体目标：(1)分别确定与生长锥吸引和排斥转向相关和/或负责的不对称运动活动；(2)分别检查与吸引和排斥相关的局部细胞骨架事件；(3)阐明微管和肌动蛋白细胞骨架的确切作用，以及它们在控制生长锥中的相互作用；(4)确定空间和时间调控的膜回收是否有助于引导生长锥转向，以及如何促进生长锥转向。长期目标是分别确定吸引和排斥转向的共同和特定的细胞事件，从而更好地理解轴突引导下的细胞机制，从而产生高度有序的大脑结构。

DESCRIPTION (provided by applicant): Formation of specific neuronal connections requires accurate guidance of developing axons to their correct targets by a variety of surface-bound and diffusible guidance cues. Axon guidance is also critically important for functional repair of damaged neuronal connections after brain injuries and diseases. During guidance, the motile growth cone at the tip of the axon senses direction from extracellular space and steers the axon through the complex environment of developing embryos to reach its specific target. Significant progress has been made in recent years towards the molecular identification of a variety of surface-bound and diffusible guidance molecules. However, the cellular mechanisms underlying directional sensing and steering of the growth cone remain largely unknown. The proposed study aims to understand how the growth cone responds to different types of diffusible guidance molecules to steer in a particular direction. The working hypothesis to be tested is as follow: different extracellular cues can act through different signaling pathways, but they all elicit localized signaling cascades that target the cytoskeleton in a spatially and temporally restricted fashion to cause the growth cone to steer in a specific direction. Taking advantage of the large size of Xenopus growth cones, an in vitro system for rigorous assay of growth cone turning, and a combination of high-resolution digital imaging and intracellular manipulation techniques, the research team plans to thoroughly investigate the cellular components associated with and, particularly, responsible for specific steering behaviors (attractive or repulsive) of the growth cone. The proposed project has four specific aims: (1) to determine the asymmetric motile activities associated with and/or responsible for growth cone attractive and repulsive turning, respectively, (2) to examine the local cytoskeletal events associated with attraction and repulsion respectively, (3) to elucidate the precise role of the microtubules and actin cytoskeleton, as well as their interactions, in steering the growth cone, and (4) to determine whether and how spatially- and temporally-regulated membrane recycling contributes to guided growth cone steering. The long-term goal is to identify the cellular events that are common as well as specific for attractive and repulsive turning, respectively, thus achieving a better understanding towards the cellular mechanisms underlying axon guidance for the generation of highly ordered brain architecture.