Elucidating mechanistic connections between guidance signaling, microtubule regulation, and growth cone steering

项目摘要/摘要 这项提议聚焦于神经元生长锥体如何被引导到它们的目标这一根本问题上。它 早就确定生长锥的导航依赖于F-肌动蛋白和肌动蛋白的调节变化 微管(MT)对引导提示的反应动力学。然而，这些线索的机制 带来具体变化的生长锥MT动态尚未得到解决。这项提议就是针对这一点的。 通过研究MT聚合酶和‘正端跟踪蛋白’(TIP)的功能，XMAP215， 以及它的监管机制。我们最近发布的数据确定了XMAP215绑定的能力 直接转化为F-肌动蛋白，并促进MT延伸进入生长锥。这与未公布的数据相辅相成 该激酶信号调节XMAP215促进MT-F-肌动蛋白相互作用的能力。我们的数据支持 XMAP215通过不同于其他结构域介导MT-F-肌动蛋白偶联的模型 调节MT聚合，以及XMAP215将来自上游指导线索的信号转换为变化 在细胞骨架的协调中，最终指导生长锥的运动。我们将在非洲爪哇进行测试，使用 一系列互补的基于细胞和生化的方法。具体目标是：目标1-确定 XMAP215与F-肌动蛋白体外相互作用机制的研究我们将确定特定的领域 与F-肌动蛋白结合的XMAP215以及识别结合是如何调节的。我们将生产各种不同的 缺失和突变的XMAP215蛋白，我们将使用一系列生化技术，包括 F-肌动蛋白/MT共沉淀结合分析、多波长TIRF显微镜和体外细胞骨架 重组分析，质谱学，以及EM实验，以剖析其机制 XMAP215在体外可与F-肌动蛋白结合。目的2-确定XMAP215与F-肌动蛋白相互作用的调节 调节培养神经元的细胞骨架动力学和生长锥行为。我们将使用结构化的 照明显微镜(SIM)、旋转圆盘共聚焦显微镜和针尖的定量成像分析 表达不同XMAP215突变体后培养神经元的动力学研究 洞察XMAP215如何在轴突生长过程中调节生长锥体中MT-F-肌动蛋白的相互作用。目标3- 定义XMAP215与F-肌动蛋白的相互作用如何有助于准确的轴突引导。我们发现， 培养的生长锥需要正常的XMAP215水平才能从轴突引导线索中排斥， 斯利特2。我们将使用体外和体内轴突引导分析相结合的方法来确定生长锥 转向受到XMAP215促进MT-F-肌动蛋白相互作用的能力的影响。这些目标的结果 将揭示引导信号之间的直接联系，是唯一具有良好特性的肌动蛋白的调节 聚合酶(XMAP215)和细胞骨架协调，在生长锥体转向的背景下。因此， 这项拟议的工作有可能将长期需要的机械性理解带入如何 细胞外信号控制细胞骨架动力学，在神经发育过程中有效地控制生长锥体。

Project Summary/Abstract This proposal focuses on the fundamental question of how neuronal growth cones are guided to their targets. It has long been established that growth cone navigation depends on regulated changes in both F-actin and microtubule (MT) dynamics in response to guidance cues. However, the mechanisms by which these cues bring about specific changes in growth cone MT dynamics are unresolved. This proposal takes aim at that void, by investigating the function of the MT polymerase and ‘plus-end tracking protein’ (+TIP), XMAP215, along with its regulatory mechanisms. Our recently published data identified an ability for XMAP215 to bind directly to F-actin and promote MT extension into the growth cone. This is complemented by unpublished data that kinase signaling modulates the ability of XMAP215 to promote MT-F-actin interaction. Our data support a model in which XMAP215 mediates MT-F-actin coupling through structural domains distinct from those regulating MT polymerization, and that XMAP215 converts signals from upstream guidance cues into changes in cytoskeletal coordination, ultimately directing growth cone motility. We will test this in Xenopus laevis using an array of complementary cell-based and biochemical approaches. The specific aims are: Aim 1 – Identify the mechanism by which XMAP215 interacts with F-actin in vitro. We will determine the specific domain of XMAP215 that binds to F-actin as well as identify how the binding is regulated. We will produce a variety of deletion and phosphomutant XMAP215 proteins, and we will use a series of biochemical techniques including F-actin/MT co-sedimentation binding assays, multi-wavelength TIRF microscopy with in vitro cytoskeletal reconstitution assays, mass spectrometry, as well as EM experiments, to dissect the mechanism by which XMAP215 binds to F-actin in vitro. Aim 2 - Determine how regulation of XMAP215 interaction with F-actin modulates cytoskeletal dynamics and growth cone behaviors in cultured neurons. We will use structured illumination microscopy (SIM), spinning disk confocal microscopy, and quantitative imaging analysis of +TIP dynamics in cultured neurons, after expressing various XMAP215 mutants, to uncover new mechanistic insights into how XMAP215 regulates MT-F-actin interactions in growth cones during axon outgrowth. Aim 3 - Define how XMAP215 interaction with F-actin contributes to accurate axon guidance. We have discovered that normal XMAP215 levels are required for cultured growth cones to be repelled from the axon guidance cue, Slit2. We will use a combination of ex vivo and in vivo axon guidance assays to determine how growth cone steering is impacted by the ability of XMAP215 to facilitate MT-F-actin interactions. The results of these Aims will reveal the direct links between guidance cue signaling, kinase regulation of the only well-characterized MT polymerase (XMAP215), and cytoskeletal coordination, within the context of growth cone steering. As such, this proposed work has the potential to bring a long-needed mechanistic understanding to the question of how extracellular cues govern cytoskeletal dynamics to effectively steer growth cones during neurodevelopment.