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了解神经发育的基本过程，也可能为诱导受损神经元的生长锥运动和再生提供策略。虽然已经取得了很大的进展，在确定生长锥的指导信号和它们的受体，相对知之甚少，这些信号是如何被转导到细胞骨架动力学的变化，所需的定向生长锥运动。然而，很明显，多个指导信号汇聚在细胞骨架上，最终必须整合到一个协调的反应中。结合并直接调节细胞骨架的蛋白质可能因此成为指导信号的最终解释者。本提案的总体目标是确定关键的生长锥细胞骨架调节蛋白，并确定其在生长锥运动和指导中的作用。实验将集中在Arp 2/3复合物，它使分支肌动蛋白丝的形成成核，并在许多类型的肌动蛋白为基础的细胞运动中发挥重要作用。最近的研究结果表明，Arp 2/3是生长锥易位的负调节因子，Arp 2/3依赖的肌动蛋白结构在协调生长锥中的肌动蛋白和微管动力学中起着重要作用。我们将测试的假设，Arp 2/3调节细胞骨架动力学响应指导信号。结合分子生物学技术、活细胞成像技术和相关的电子显微镜技术，将用于推断Arp 2/3在生长锥运动和寻路中的作用机制。Arp 2/3的组织特异性抑制将用于表征Arp 2/3在发育神经系统中的作用。最后，在生长锥中激活Arp 2/3的蛋白质的鉴定将提供深入了解Arp 2/3是如何通过上游信号通路调节的。

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.