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

阐明引导信号传导、微管调节和生长锥转向之间的机制联系

• 批准号: 10362374
• 负责人: Laura Anne LOWERY
• 金额: $ 44.85万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10362374
• 关键词: Address; Area; Award; Axon; Behavior; Binding; Biochemical; Biological Assay; Brain; Cells; Complement; Confocal Microscopy; Coupling; Cues; Data; Development; Disease; Elements; F-Actin; Future; Growth Cones; In Vitro; Lead; Letters; Lighting; Link; Mass Spectrum Analysis; Mediating; Mental disorders; Microscopy; Microtubule Polymerization; Microtubules; Modeling; Molecular; Morphology; Nervous System Physiology; Neurons; Phosphotransferases; Play; Polymerase; Positioning Attribute; Prevention strategy; Proteins; Publishing; Regulation; Role; Schizophrenia; Sedimentation process; Series; Signal Pathway; Signal Transduction; Structure; Techniques; Testing; Time; Total Internal Reflection Fluorescent; Work; Xenopus laevis; autism spectrum disorder; axon guidance; base; cell motility; design; experience; experimental study; extracellular; in vivo; insight; microscopic imaging; mutant; neurodevelopment; neuronal growth; neuropsychiatric disorder; novel; public health relevance; quantitative imaging; reconstitution; relating to nervous system; response; tool; treatment strategy

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 This is complemented by unpublished data that kinase signaling modulates the ability of XMAP215 to promote MT-F-actin interaction.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

项目摘要/摘要 该提案着重于神经元生长锥如何指导其目标的基本问题。 长期以来一直确定，生长锥导航取决于F-肌动蛋白和 微管（MT）动力学响应指导线索。但是，这些提示的机制 带来生长锥体MT动力学的特定变化。该提议的目标是 通过研究MT聚合酶和“加末端跟踪蛋白”（+TIP）的功能，XMAP215， 及其监管机制。我们最近发布的数据确定了XMAP215绑定的能力 直接进行F-肌动蛋白并促进MT扩展到生长锥。这是由未发表的数据完成的 这种激酶信号传导调节XMAP215促进MT-F-肌动蛋白相互作用的能力。我们的数据支持 XMAP215介导MT-F-肌动蛋白偶联的模型通过与之不同的结构域 调节MT聚合，并且XMAP215将信号从上游指导提示转换为变化 在细胞骨架配位中，最终导致生长锥运动。我们将在Xenopus laevis中使用 一系列基于互补的细胞和生化方法。具体目的是：目标1 - 确定 XMAP215在体外与F-肌动作相互作用的机制。我们将确定 XMAP215与F-肌动蛋白结合并确定如何调节结合。我们将产生各种各样的 缺失和磷化剂XMAP215蛋白质，我们将使用一系列的生化技术 F-肌动蛋白/MT共同补习结合测定，多波长TIRF显微镜与体外细胞骨架 重建测定法，质谱和EM实验，以剖析该机制 XMAP215在体外与F-肌动蛋白结合。 AIM 2-确定XMAP215的调节如何与F-肌动蛋白相互作用 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），旋转磁盘共聚焦显微镜和 +尖端的定量成像分析 表达各种XMAP215突变体后，培养的神经元中的动力学，以发现新的机械 洞察XMAP215如何调节轴突生长过程中生长锥中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指导。我们发现 培养的生长锥从轴突引导提示中排斥，需要正常的XMAP215水平 SLIT2。我们将结合体内和体内轴突指导测定法来确定生长锥如何 转向受XMAP215促进MT-F-肌动相互作用的能力的影响。这些目标的结果 将揭示指导提示信号传导，唯一特征良好的MT的激酶调节之间的直接联系 在生长锥转向的背景下，聚合酶（XMAP215）和细胞骨架配位。像这样， 这项提出的工作有可能将长期以来的机械理解带入有关如何 细胞外提示控制细胞骨架动力学，以在神经发育过程中有效茎生长锥。