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将发育中的轴突准确引导到其正确的靶点。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，轴突尖端的运动生长锥感知来自细胞外空间的方向，并引导轴突穿过发育胚胎的复杂环境以到达其特定目标。近年来，对各种表面结合和可扩散的导向分子的分子鉴定取得了重大进展。然而，生长锥的定向感知和转向的细胞机制在很大程度上仍然未知。这项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，以引导特定方向。待检验的工作假设如下：不同的细胞外信号可以通过不同的信号通路起作用，但它们都引发局部信号级联，以空间和时间受限的方式靶向细胞骨架，以使生长锥转向特定方向。利用非洲爪蟾生长锥的大尺寸，用于生长锥转向的严格测定的体外系统，以及高分辨率数字成像和细胞内操作技术的组合，研究小组计划彻底调查与生长锥相关的细胞成分，特别是负责特定的转向行为（吸引或排斥）。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动相关和/或负责生长锥吸引和排斥转动的不对称运动活动，（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精确作用，以及它们的相互作用，在操纵生长锥中，以及（4）确定空间和时间调节的膜再循环是否以及如何有助于引导生长锥转向。长期的目标是确定细胞的事件，是共同的，以及具体的吸引力和排斥性的转向，分别，从而实现对细胞机制的更好的理解轴突指导下产生高度有序的大脑架构。

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.