Mechanism of a Microtubule Polymerase Complex Critical for Axon Outgrowth

微管聚合酶复合物对轴突生长至关重要的机制

基本信息

  • 批准号:
    10043009
  • 负责人:
  • 金额:
    $ 15.55万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2020
  • 资助国家:
    美国
  • 起止时间:
    2020-06-01 至 2024-05-31
  • 项目状态:
    已结题

项目摘要

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.
在解释微管如何在神经元中活动方面,我们的知识存在根本性的空白。的 微管细胞骨架对于神经元结构、动力学和运输是至关重要的。空间因素如何 调节微管细胞骨架以促进轴突生长的机制还知之甚少。微管 聚合酶是定位于微管正末端并促进微管聚合的关键调节剂。 在神经元中,微管聚合酶与Sentin和TACC因子相关。敲低 微管聚合酶或其相关因子损害微管动力学,产生异常的 对引导线索的反应和轴突生长的缺陷。微管聚合酶如何结合Sentin, TACC以及这种复合物如何共同调节微管细胞骨架还知之甚少。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, 确定该保守结构域具有微管结合活性。根据初步数据,我们 假设Sentin-Msps-TACC微管聚合酶复合物的组分起作用, 协同地结合微管晶格并驱动微管聚合。的理由 拟议的研究是确定微管聚合酶复合物的成分如何增强 微管聚合是神经元结构、极性、信号和动力学所需的聚合物。两 具体目的是研究微管聚合酶复合物的结构和机制。第一个目标是 使用X射线晶体学确定微管聚合酶复合物的分子结构, 阐明结合化学计量和亲和力,设计和测试防止复合物形成的突变。的 第二个目的是绘制微管聚合酶复合物中参与微管结合的残基, 使用体外重构微管动力学测定复合物如何影响微管动力学 比色法这两个独立的目标是建立一个高分辨率的Sentin-Msps-TACC模型 微管聚合酶活性。这种方法是创新的,因为它汇集了一套不同的生物物理, 生物化学和细胞分析,以提供微管聚合酶的多分辨率模型 机制这项拟议中的研究意义重大,因为它测试了一个核心分子过程, 神经元的形状、动力学和信号传导。调查的长期目标是确定 微管聚合酶复合物调节神经生长锥细胞骨架动力学 引导线索拟议的研究将通过建立一个机制框架, 可以研究轴突生长中基于细胞间隙的缺陷, 突变的微管聚合酶、Sentin和TACC表型,包括自发性轴突收缩和 对轴突引导线索的反应缺陷。

项目成果

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Kevin C Slep其他文献

Kevin C Slep的其他文献

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{{ truncateString('Kevin C Slep', 18)}}的其他基金

Molecular Mechanisms of Spectraplakins
Spectraplakins 的分子机制
  • 批准号:
    9118250
  • 财政年份:
    2015
  • 资助金额:
    $ 15.55万
  • 项目类别:
Regulators of Centriole Duplication and Structure
中心粒复制和结构的调节因子
  • 批准号:
    8059658
  • 财政年份:
    2010
  • 资助金额:
    $ 15.55万
  • 项目类别:
Regulators of Centriole Duplication and Structure
中心粒复制和结构的调节因子
  • 批准号:
    7875565
  • 财政年份:
    2010
  • 资助金额:
    $ 15.55万
  • 项目类别:

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