Mechanism and Coordination of Cytoplasmic Dynein Motility

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，运动蛋白和细胞质动力蛋白，可以沿着微管连续进行许多步，以长距离运输货物。这种连续的运动，称为加工性，需要两个运动域之间的协调，以防止微管过早释放。详细的结构和机制模型存在的动力蛋白，但机制和协调的动力蛋白的运动仍然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与其他细胞骨架马达具有不同的机械特征。动力蛋白形成一个大的多亚基复合物，其核心由一个AAA atp酶结构域环组成。环内ATP水解驱动的构象变化是动力蛋白产生和运动的基础。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。在我们的前期工作中，我们利用酿酒葡萄球菌表达重组动力蛋白马达，并在体外表征了动力蛋白的步进行为。在这一建议中，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周期并相互相对移动。不同的AAA结构域的具体作用将通过选择性地突变一个环上的atp酶位点来研究。其次，我们将研究atp驱动的马达结构域的构象状态如何驱动动力蛋白的动力行程并改变微管结合亲和力。在动力行走中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被控制，直到另一个头部完成其前进的步骤。第三，我们将建立dynein负端方向性的结构基础。总之，我们提出的研究代表了动力蛋白在单分子水平上的构象和化学状态的重点研究，因为活性动力蛋白二聚体沿着表面固定化的mt移动。我们希望显著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。

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.