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

项目概要/摘要 这项建议的重点是神经生长锥是如何引导到他们的目标的基本问题。它 长期以来，生长锥导航依赖于F-肌动蛋白和 微管（MT）动态响应的指导线索。然而，这些线索的机制 引起生长锥MT动力学的具体变化尚未解决。这项建议的目的是， 空，通过研究MT聚合酶和“+末端跟踪蛋白”（+TIP），XMAP 215， 沿着其调节机制。我们最近发表的数据确定了XMAP 215结合 直接与F-肌动蛋白结合并促进MT延伸到生长锥中。这是由未公布的数据补充 激酶信号调节XMAP 215促进MT-F-肌动蛋白相互作用的能力。我们的数据支持 模型，其中XMAP 215通过与那些结构域不同的结构域介导MT-F-肌动蛋白偶联 调节MT聚合，并且XMAP 215将来自上游指导线索的信号转化为变化， 在细胞骨架协调，最终指导生长锥运动。我们将在非洲爪蟾身上测试， 一系列互补的基于细胞和生物化学的方法。具体目标如下：目标1 -确定 XMAP 215与F-actin在体外相互作用的机制。我们将确定的具体领域 XMAP 215结合F-肌动蛋白以及确定如何调节结合。我们将生产各种 缺失和磷酸突变XMAP 215蛋白，我们将使用一系列生化技术，包括 F-肌动蛋白/MT共沉降结合试验，多波长TIRF显微镜，体外细胞骨架 重组分析，质谱分析，以及EM实验，剖析机制， XMAP 215在体外与F-肌动蛋白结合。目的2 -确定XMAP 215与F-肌动蛋白相互作用的调节 调节培养神经元中的细胞骨架动力学和生长锥行为。我们将使用结构化 照明显微镜（SIM）、旋转圆盘共聚焦显微镜和+TIP的定量成像分析 在表达各种XMAP 215突变体后，培养的神经元中的动力学，以揭示新的机制。 深入了解XMAP 215如何调节轴突生长过程中生长锥中MT-F-肌动蛋白相互作用。目标3 - 定义XMAP 215与F-actin的相互作用如何有助于精确的轴突引导。我们发现 正常的XMAP 215水平是培养的生长锥排斥轴突导向因子所必需的， 狭缝2。我们将使用离体和体内轴突导向试验的组合来确定生长锥如何在体内生长。 操纵受到XMAP 215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 将揭示引导信号传导、激酶调节之间的直接联系， 聚合酶（XMAP 215）和细胞骨架协调，在生长锥转向的背景下。因此，在本发明中， 这项拟议中的工作有可能为如何实现这一目标的问题带来一种长期需要的机械理解 细胞外线索支配细胞骨架动力学，以在神经发育期间有效地操纵生长锥。

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 This is complemented by unpublished data that kinase signaling modulates the ability of XMAP215 to promote MT-F-actin interaction.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.