Cytoskeletal Signaling and Axon Guidance

摘要 轴突末端的迁移生长锥感知并响应引导信息。生长锥显示动态板状伪足和丝状伪足突起，参与生长和指导。 这些研究的目的是使用C。elegans了解细胞骨架的信号网络，连接轴突的指导信号的变化，生长锥形态和通过肌动蛋白细胞骨架生长。上一个资助期的结果表明，三个肌动蛋白调节系统，Arp 2/3，ARP-115/abLIM和ARP-34/Enabled，冗余地控制轴突寻路。Arp2/3复合物和β-115/abLIM在Rac GTP酶的下游冗余地起作用，并与β-34/Enabled平行。这些分子中的每一个控制生长锥丝状伪足的形成，可能解释突变体中轴突寻路缺陷。多个肌动蛋白调节系统有助于轴突导向和生长锥丝状伪足的形成，这解释了在轴突导向研究中经常观察到的遗传冗余。为了了解生长锥中调节肌动蛋白结构的分子事件，重要的是要了解这些单独的肌动蛋白调节网络如何发挥作用，以及它们如何在生长锥生长和指导中相互作用。在这个建议中，遗传，分子和体内细胞成像方法将被用来解剖生长锥形态和生长的细胞骨架信号通路的作用。许多途径和分子已被确定影响轴突寻路。这些实验超越了“逐个基因”的方法来研究轴突寻路，而是旨在了解分子如何在通路和网络中相互关联，以控制轴突寻路和生长锥形态。为了补充遗传分析，体内细胞成像研究旨在了解发育背景下生长锥形态发生的遗传冗余途径的独特或重叠贡献。该提案的重点是CDC-42 GTP酶在轴突寻路中作用于Rac GTP酶上游的想法。 提出了表征调控CDC-42的分子和可能将CDC-42与Rac信号传导联系起来的分子的实验。第一个和第二个目标旨在测试CDC-42在轴突寻路中作用于Rac GTP酶上游的想法。第三个目的是探测活化C激酶的NIK-15激酶和RACK-1/受体的作用以及它们在Rac非依赖性途径中与CDC-42下游的NIK-115/abLIM的相互作用。第四个目标整合了前三个，是表征这些途径对生长锥丝状伪足的形成和生长过程中的形态的影响。这些实验的结果将显着有助于理解的细胞骨架信号网络参与生长锥形态的目标，并将开始，以解决这些不同的途径在轴突发育的细胞作用。

Abstract The migrating growth cone at the distal axon tip senses and responds to guidance information. Growth cones display dynamic lamellipodial and filopodial protrusions that are involved in outgrowth and guidance. The aim of these studies is to use C. elegans to understand the cytoskeletal signaling networks that link axon guidance signals to changes in growth cone morphology and outgrowth via the actin cytoskeleton. Results from the previous grant period showed that three actin-regulatory systems, Arp2/3, UNC-115/abLIM, and UNC-34/Enabled, redundantly control axon pathfinding. The Arp2/3 complex and UNC-115/abLIM act redundantly downstream of Rac GTPases and in parallel to UNC-34/Enabled. Each of these molecules controlled the formation of growth cone filopodia, possibly explaining axon pathfinding defects in mutants. That multiple actin regulatory systems contribute to axon guidance and the formation of growth cone filopodia explains the genetic redundancy often observed in studies of axon guidance. In order to understand the molecular events in the growth cone that regulate actin structure, it will be important to understand how these individual actin regulatory networks function and how they interact in growth cone outgrowth and guidance. In this proposal, genetic, molecular, and in vivo cellular imaging approaches will be used to dissect the roles of cytoskeletal signaling pathways in growth cone morphology and outgrowth. Many pathways and molecules have been identified that affect axon pathfinding. These experiments move beyond the "gene by gene" approach to studying axon pathfinding and instead are designed to understand how molecules relate to one another in pathways and networks to control axon pathfinding and growth cone morphology. To complement the genetic analyses, in vivo cellular imaging studies are designed to understand the unique or overlapping contributions of the often genetically-redundant pathways to growth cone morphogenesis in a developmental context. The focus of the proposal is the idea that the CDC-42 GTPase acts upstream of Rac GTPases in axon pathfinding. Experiments to characterize molecules that regulate CDC-42 and those that might link CDC-42 to Rac signaling are proposed. The first and second aims aim test the idea that CDC-42 acts upstream of Rac GTPases in axon pathfinding. The third aim is to probe the role of the MIG-15 NIK kinase and RACK-1/Receptor for activated C kinase and their interaction with UNC-115/abLIM downstream of CDC-42 in a Rac-independent pathway. The fourth aim integrates the first three and is to characterize the effects of these pathways on growth cone filopodia formation and morphology during outgrowth. The results of these experiments will significantly contribute to the goal of understanding the cytoskeletal signaling networks involved in growth cone morphology and will begin to address the cellular roles of these distinct pathways in axon development.