Mechanism and Coordination of Cytoplasmic Dynein Motility

细胞质动力蛋白运动的机制和协调

基本信息

  • 批准号:
    8108831
  • 负责人:
  • 金额:
    $ 28.59万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-04-01 至 2016-03-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Molecular motors drive key biological processes such as intracellular cargo transport and cell division. Two dimeric motors, kinesin and cytoplasmic dynein, can take many consecutive steps along microtubules to transport cargos over long distances. This continuous movement, termed processivity, requires coordination between the two motor domains to prevent premature release from the microtubule. Detailed structural and mechanistic models exist for kinesin, but the mechanism and coordination of dynein motility remains largely unknown. Dynein's unconventional structure and distinct origin suggest that it has different mechanistic features than other cytoskeletal motors. Dynein forms a large multisubunit complex, the core of which consists of a ring of AAA ATPase domains. Conformational changes driven by ATP hydrolysis within the ring underlie dynein force generation and motion. Recent structural and biochemical studies have identified the major conformational states of monomeric dynein constructs. However, studies of active dynein dimers are lacking. As a result, the molecular basis by which ATP driven structural changes lead to unidirectional motion of a dimer as a whole is unknown. In our preliminary work, we have used S. cerevisiae to express recombinant dynein motors and characterized dynein stepping behavior in vitro. In this proposal, using single-molecule imaging methods, we propose to dissect the coordination between the nucleotide and conformational states of the motor domains in native and engineered dynein constructs. We have three specific aims. First, using multicolor tracking methods, we will directly observe how the AAA ring domains coordinate their nucleotide cycles and move relative to each other. The specific roles of distinct AAA domains will be studied by selectively mutating out the ATPase sites in one ring. Second, we will investigate how ATP-driven conformational states of the motor domain drive the dynein powerstroke and alter microtubule-binding affinity. The ability to perform these measurements as dynein walks will allow us to demonstrate whether the mechanical cycle of one head is gated until the other head completes its forward step. Third, we will establish the structural basis of dynein's minus-end directionality. Together, our proposed research represents a focused investigation of the conformational and chemical states of dynein at a single-molecule level, as active dynein dimers move along surface-immobilized MTs. We hope to significantly advance understanding of dynein's fundamental mechanochemistry and learn how it achieves retrograde transport of intracellular cargos. PUBLIC HEALTH RELEVANCE: Consistent with its fundamental roles in neurobiology and cell development, complete knockouts of dynein stop the entire microtubule transport machinery and inhibit mitosis. Mutations that alter the processivity or velocity of dynein movement lead to pathogenesis of motor neuron degeneration, including the Alzheimer's disease and ALS. Detailed studies of dynein-related diseases require replacement of engineered dynein mutants whose motility properties have been altered in predictable ways. Dissecting the mechanism of dynein motility is a prerequisite of understanding the molecular basis of these diseases.
描述(申请人提供):分子马达驱动关键的生物过程,如细胞内货物运输和细胞分裂。两个二聚体马达,激动素和细胞质动力蛋白,可以沿着微管连续走很多步,以远距离运输货物。这种连续的运动称为过程性,需要两个运动域之间的协调,以防止从微管过早释放。目前已有关于动力蛋白的详细结构和机制模型,但动力蛋白运动的机制和协调在很大程度上仍不清楚。动力蛋白的非常规结构和独特的起源表明,它具有不同于其他细胞骨架马达的机械特性。动力蛋白形成一个大的多亚单位复合体,其核心由一环的AAA ATPase结构域组成。环内ATP水解所驱动的构象变化是动力产生和运动的基础。最近的结构和生化研究已经确定了单体动力蛋白结构的主要构象状态。然而,对活性动力蛋白二聚体的研究还很缺乏。因此,ATP驱动的结构变化导致二聚体作为一个整体单向运动的分子基础是未知的。在我们的前期工作中,我们利用酿酒酵母表达了重组动力蛋白马达,并在体外表征了动力蛋白的步进行为。在这项建议中,使用单分子成像方法,我们建议剖析天然和工程动力蛋白结构中运动域的核苷酸和构象状态之间的协调。我们有三个具体目标。首先,使用多色跟踪方法,我们将直接观察AAA环结构域是如何协调它们的核苷酸环以及如何相互移动的。不同的AAA结构域的具体作用将通过选择性地突变一个环中的ATPase位点来研究。其次,我们将研究ATP驱动的运动域的构象状态如何驱动动力蛋白PowerStroke和改变微管结合的亲和力。在动力蛋白行走时进行这些测量的能力将使我们能够证明一个头部的机械循环是否处于门控状态,直到另一个头部完成其前进步骤。第三,我们将建立动力蛋白负端方向性的结构基础。总之,我们提出的研究是在单分子水平上集中研究动力蛋白的构象和化学状态,当活性动力蛋白二聚体沿着表面固定的MTS移动时。我们希望显著提高对动力蛋白基本机械力化学的理解,并了解它如何实现细胞内货物的逆行运输。 与公共健康相关:与其在神经生物学和细胞发育中的基本作用一致,动力蛋白的完全敲除停止了整个微管运输机制,并抑制了有丝分裂。改变动力蛋白运动过程或速度的突变会导致运动神经元变性,包括阿尔茨海默病和肌萎缩侧索硬化症。对动力蛋白相关疾病的详细研究需要更换动力蛋白工程突变体,这些突变体的运动性已以可预测的方式改变。剖析动力蛋白运动的机制是了解这些疾病分子基础的前提。

项目成果

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Ahmet Yildiz其他文献

Ahmet Yildiz的其他文献

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{{ truncateString('Ahmet Yildiz', 18)}}的其他基金

The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins
细胞质和纤毛动力蛋白的机制和调控
  • 批准号:
    10133096
  • 财政年份:
    2020
  • 资助金额:
    $ 28.59万
  • 项目类别:
The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins
细胞质和纤毛动力蛋白的机制和调控
  • 批准号:
    10594962
  • 财政年份:
    2020
  • 资助金额:
    $ 28.59万
  • 项目类别:
The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins
细胞质和纤毛动力蛋白的机制和调控
  • 批准号:
    10378048
  • 财政年份:
    2020
  • 资助金额:
    $ 28.59万
  • 项目类别:
Structural and Functional Characterization of Telomere Protection and Maintenance
端粒保护和维持的结构和功能表征
  • 批准号:
    9262255
  • 财政年份:
    2016
  • 资助金额:
    $ 28.59万
  • 项目类别:
Structural and Functional Characterization of Telomere Protection and Maintenance
端粒保护和维持的结构和功能表征
  • 批准号:
    9083326
  • 财政年份:
    2016
  • 资助金额:
    $ 28.59万
  • 项目类别:
Mechanism and Coordination of Cytoplasmic Dynein Motility
细胞质动力蛋白运动的机制和协调
  • 批准号:
    8242076
  • 财政年份:
    2011
  • 资助金额:
    $ 28.59万
  • 项目类别:
Understanding the Mechanism and Regulation of the Human Cytoplasmic Dynein Complex
了解人类细胞质动力蛋白复合物的机制和调节
  • 批准号:
    9267494
  • 财政年份:
    2011
  • 资助金额:
    $ 28.59万
  • 项目类别:
Mechanism and Coordination of Cytoplasmic Dynein Motility
细胞质动力蛋白运动的机制和协调
  • 批准号:
    8641392
  • 财政年份:
    2011
  • 资助金额:
    $ 28.59万
  • 项目类别:
Mechanism and Coordination of Cytoplasmic Dynein Motility
细胞质动力蛋白运动的机制和协调
  • 批准号:
    8450779
  • 财政年份:
    2011
  • 资助金额:
    $ 28.59万
  • 项目类别:
Mechanism and Coordination of Cytoplasmic Dynein Motility
细胞质动力蛋白运动的机制和协调
  • 批准号:
    8865640
  • 财政年份:
    2011
  • 资助金额:
    $ 28.59万
  • 项目类别:
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