Cytoskeletal Mechanisms of Growth Cone Motility

描述(由申请人提供)： 了解生长锥运动的方式和原因将有助于阐明神经发育的基本过程，也可能提出诱导受损神经元的生长锥运动和再生的策略。虽然在识别生长锥引导信号及其受体方面已经取得了很大进展，但对于这些信号如何传导到细胞骨架动力学的变化，这是定向生长锥运动所必需的，人们相对知之甚少。然而，很明显，多个引导信号会聚在细胞骨架上，最终必须整合成一个协调的反应。因此，与细胞骨架结合并直接调节细胞骨架的蛋白质可能是引导信号的最终解释者。这项提案的总体目标是确定关键的生长锥细胞骨架调节蛋白，并确定它们在生长锥运动和引导中的作用。实验将集中在Arp2/3复合体上，它核化分支肌动蛋白细丝的形成，并在许多类型的基于肌动蛋白的细胞运动中发挥重要作用。最近的研究结果表明，Arp2/3是生长锥移位的负调控因子，依赖Arp2/3的肌动蛋白结构在协调生长锥内肌动蛋白和微管动力学方面发挥着重要作用。我们将测试Arp2/3调节细胞骨架动力学以响应引导信号的假设。结合分子和生化技术、活细胞成像和相关的电子显微镜，将推测Arp2/3在生长锥运动和通路中的作用机制。Arp2/3的组织特异性抑制将被用来表征Arp2/3在神经系统发育中的作用。最后，识别激活生长锥中Arp2/3的蛋白质将有助于深入了解Arp2/3是如何受上游信号通路调控的。

DESCRIPTION (provided by applicant): Understanding how and why the growth cone moves will shed light on the fundamental processes of neurodevelopment and may also suggest strategies to induce growth cone motility and regeneration in damaged neurons. While much progress has been made in identifying growth cone guidance signals and their receptors, relatively little is known about how these signals are transduced into the changes in cytoskeletal dynamics that are required for directed growth cone motility. It is clear, however, that multiple guidance signals converge on the cytoskeleton and must ultimately be integrated into a coordinated response. Proteins that bind to and directly regulate the cytoskeleton may thus serve as the ultimate interpreters of guidance signals. The overall goal of this proposal is to identify key growth cone cytoskeletal regulatory proteins and determine their role in growth cone motility and guidance. Experiments will focus on the Arp2/3 complex, which nucleates the formation of branched actin filaments and plays an essential role in many types of actin-based cell motility. Recent findings indicate that Arp2/3 is a negative regulator of growth cone translocation and that Arp2/3-dependent actin structures play an important role in coordinating actin and microtubule dynamics in the growth cone. We will test the hypothesis that Arp2/3 regulates cytoskeletal dynamics in response to guidance signals. A combination of molecular and biochemical techniques, live cell imaging and correlative electron microscopy will be used to deduce the mechanism of Arp2/3 function in growth cone motility and pathfinding. Tissue specific inhibition of Arp2/3 will be used to characterize the role of Arp2/3 in the developing nervous system. Finally, identification of the proteins that activate Arp2/3 in growth cones will provide insight into how Arp2/3 is regulated by upstream signaling pathways.