Mechanism of a Microtubule Polymerase Complex Critical for Axon Outgrowth

在解释神经元中微管是如何聚合的方面，我们的知识存在一个根本的空白。这个 微管细胞骨架对神经元的结构、动力学和运输至关重要。空间因素如何 调节微管细胞骨架以促进轴突的生长还知之甚少。微管 聚合酶是定位于微管+末端并促进微管聚合的关键调节因子。 在神经元中，微管聚合酶与Sentin和TACC因子有关。被击倒的 微管聚合酶或其相关因子损害微管动力学，产生异常 轴突生长中对引导线索和缺陷的反应。微管聚合酶如何与感觉素和 TACC以及这个复合体是如何共同调节微管细胞骨架的还知之甚少。我们有 将Sentin和TACC结合到微管聚合酶MSPs的保守C末端结构域，并 确定该保守结构域具有微管结合活性。根据我们的初步数据，我们 假设Sentin-MSPs-TACC微管聚合酶复合体的组成起作用 协同结合微管晶格，推动微管聚合。该计划的基本原理 拟议的研究是确定微管聚合酶复合体的成分如何增强 微管聚合，一种神经结构、极性、信号和动力学所需的聚合物。二 特定的目的是研究微管聚合酶复合体的结构和机制。第一个目标是 用X射线结晶学确定微管聚合酶复合体的分子结构， 阐明结合化学计量学和亲和力，并设计和测试防止复合体形成的突变。这个 第二个目标是定位参与微管结合和微管结合的微管聚合酶复合体中的残基。 用体外重组微管动力学研究该复合体对微管动力学的影响 化验。这两个独立的目标致力于开发Sentin-MSPs-TAcc的高分辨率模型 微管聚合酶活性。这种方法是创新的，因为它汇集了一组不同的生物物理， 提供微管聚合酶多分辨模型的生化和细胞分析 机制。这项拟议的研究意义重大，因为它测试了 神经元的形状、动力学和信号。调查的长期目标是确定 微管聚合酶复合体调节神经元生长锥体细胞骨架动力学 引导线索。拟议的研究将通过建立一个机制框架来影响公共健康 在轴突生长中，可以研究哪些基于细胞骨架的缺陷，从而提供对 突变的微管聚合酶、Sentin和TACC表型，包括自发的轴突回缩和 对轴突引导线索的反应有缺陷。

There is a fundamental gap in our knowledge to explain how microtubules polymerize in neurons. The microtubule cytoskeleton is critical for neuronal structure, dynamics, and trafficking. How factors spatially regulate the microtubule cytoskeleton to promote axon outgrowth is poorly understood. Microtubule polymerases are key regulators that localize to microtubule plus ends and promote microtubule polymerization. In neurons, microtubule polymerases associate with the factors Sentin and TACC. Knockdown of the microtubule polymerase or its associated factors compromises microtubule dynamics, yielding aberrant responses to guidance cues and defects in axon outgrowth. How the microtubule polymerase binds Sentin and TACC and how this complex collectively regulates the microtubule cytoskeleton is poorly understood. We have mapped Sentin and TACC binding to a conserved C-terminal domain of the microtubule polymerase Msps and determined that this conserved domain has microtubule-binding activity. Drawn from our preliminary data, we hypothesize that the components of the Sentin-Msps-TACC microtubule polymerase complex work synergistically to bind the microtubule lattice and drive microtubule polymerization. The rationale for the proposed research is to determine how the components of a microtubule polymerase complex potentiate microtubule polymerization, a polymer required for neuronal structure, polarity, signaling and dynamics. Two specific aims examine the structure and mechanism of the microtubule polymerase complex. The first aim is to determine the molecular architecture of the microtubule polymerase complex using X-ray crystallography, elucidate binding stoichiometry and affinity, and design and test mutations that prevent complex formation. The second aim is to map residues in the microtubule polymerase complex involved in microtubule binding and determine how the complex affects microtubule dynamics using an in vitro reconstituted microtubule dynamics assay. These two independent aims work to develop a high-resolution model for Sentin-Msps-TACC microtubule polymerase activity. The approach is innovative because it marshals a diverse set of biophysical, biochemical, and cellular assays to provide a multi-resolution model of the microtubule polymerase mechanism. The proposed research is significant because it tests a core molecular process required for neuronal shape, dynamics, and signaling. The investigation's long-term objective is to determine how the microtubule polymerase complex regulates cytoskeletal dynamics in neuronal growth cones in response to guidance cues. The proposed research will impact public health by establishing a mechanistic framework from which cytoskeletal-based defects in axon outgrowth can be investigated, providing molecular insight into mutant microtubule polymerase, Sentin, and TACC phenotypes, including spontaneous axon retraction and defects in the response to axon guidance cues.