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
• 负责人: Ahmet Yildiz
• 金额: $ 28.32万
• 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/8865640
• 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; Health; 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; Recombinants; Registries; Regulation; Relative (related person); Research; 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; retrograde transport; single molecule; temporal measurement; 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.

描述（由申请人提供）： 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，驱动蛋白和细胞质动力蛋白，可以采取许多连续的步骤沿着微管运输货物长距离。这种连续的运动，称为持续性，需要两个运动域之间的协调，以防止从微管过早释放。存在详细的结构和机制模型驱动蛋白，但动力蛋白运动的机制和协调仍然在很大程度上是未知的。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征。 动力蛋白形成一个大的多亚基复合物，其核心由AAA ATP酶结构域的环组成。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结构的主要构象状态。然而，缺乏对动力蛋白二聚体活性的研究。因此，ATP驱动的结构变化导致二聚体整体单向运动的分子基础是未知的。在我们的初步工作中，我们使用了S。cerevisiae中表达重组动力蛋白马达并表征动力蛋白的体外步进行为。在这个建议中，使用单分子成像方法，我们建议解剖天然和工程动力蛋白构建体中的马达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，使用荧光跟踪方法，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结构域的特定作用。其次，我们将研究如何ATP驱动的构象状态的电机域驱动动力蛋白powerstroke和改变微管结合亲和力。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周期是否被门控，直到另一个头部完成其向前的一步。第三，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的动力蛋白的构象和化学状态的集中调查，作为活性动力蛋白二聚体移动沿着表面固定的MT。我们希望能大大推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。