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
• 负责人: Ahmet Yildiz
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8242076
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Affect; Affinity; Alzheimer' s Disease; Axonal Transport; Behavior; Binding; Biochemical; Biological Process; Cell division; Cell physiology; Cells; Cellular biology; Chemicals; Complex; Development; Disease; Dynein ATPase; Elements; Engineering; Eukaryotic Cell; Event; Fluorescence Polarization; Generations; Genetic; Head; Image; In Vitro; Investigation; Kinesin; Knock-out; Lead; Learning; Measurement; Measures; Mechanics; Methods; Microtubules; Mitosis; Mitotic spindle; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motion; Motor; Movement; Mutate; Mutation; Neurobiology; Nucleotides; Pathogenesis; Peptides; Play; Positioning Attribute; Property; Proteins; Public Health; Recombinants; Registries; Regulation; Relative (related person); Research; Resolution; Role; Saccharomyces cerevisiae; Site; Structure; Surface; System; Techniques; Testing; Vesicle; Walking; Work; Yeasts; base; cell motility; controlled release; dimer; imaging modality; millisecond; motor neuron degeneration; mutant; optical imaging; optical traps; premature; prevent; public health relevance; retrograde transport; single molecule; tool; trafficking

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（由申请人提供）：分子马达驱动关键的生物过程，例如细胞内货物运输和细胞分裂。两个二聚体马达，驱动蛋白和细胞质动力蛋白，可以沿着微管采取许多连续的步骤来长距离运输货物。这种连续运动被称为持续性，需要两个运动域之间的协调，以防止微管过早释放。驱动蛋白存在详细的结构和机制模型，但动力蛋白运动的机制和协调仍然很大程度上未知。动力蛋白的非常规结构和独特的起源表明它具有与其他细胞骨架马达不同的机械特征。 动力蛋白形成一个大型多亚基复合物，其核心由 AAA ATP 酶结构域环组成。由环内 ATP 水解驱动的构象变化是动力蛋白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构建体的主要构象状态。然而，缺乏对活性动力蛋白二聚体的研究。因此，ATP 驱动结构变化导致二聚体整体单向运动的分子基础尚不清楚。在我们的前期工作中，我们使用酿酒酵母表达重组动力蛋白马达，并在体外表征了动力蛋白步进行为。在本提案中，我们建议使用单分子成像方法来剖析天然和工程动力蛋白构建体中运动结构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环结构域如何协调其核苷酸循环并相对于彼此移动。将通过选择性突变一个环中的 ATP 酶位点来研究不同 AAA 结构域的具体作用。其次，我们将研究 ATP 驱动的运动域构象状态如何驱动动力蛋白动力冲程并改变微管结合亲和力。当动力蛋白行走时执行这些测量的能力将使我们能够证明一个头的机械循环是否被门控，直到另一个头完成其前进步骤。第三，我们将建立动力蛋白负端方向性的结构基础。总之，我们提出的研究代表了在单分子水平上对动力蛋白构象和化学状态的集中研究，因为活性动力蛋白二聚体沿着表面固定的 MT 移动。我们希望显着增进对动力蛋白基本机械化学的理解，并了解它如何实现细胞内货物的逆行运输。 公众健康相关性：与动力蛋白在神经生物学和细胞发育中的基本作用相一致，动力蛋白的完全敲除会停止整个微管运输机制并抑制有丝分裂。改变动力蛋白运动的持续性或速度的突变会导致运动神经元变性的发病机制，包括阿尔茨海默病和 ALS。对动力蛋白相关疾病的详细研究需要替换工程动力蛋白突变体，其运动特性已以可预测的方式发生改变。剖析动力蛋白运动机制是了解这些疾病分子基础的先决条件。