Regulation of Cytoplasmic Dynein Based Vesicle Transport

描述（由申请人提供）：电池使用基于微管的电机，具有广泛的功能。一些马达受到外部因素的调节，这些外部因素将它们定向到不同的货物或增强或抑制酶的活性。其中一个因素是动力蛋白，这是一种高度保守的多蛋白复合物，在真核生物中普遍存在。动力蛋白以其对细胞质动力蛋白功能的贡献而闻名，但似乎也与另一种马达运动蛋白-2一起工作。在本提案中，我描述了解决两个重要问题的工作：dynactin如何与这两个马达相互作用以允许协调的双向运动，以及dynactin功能如何随着细胞周期的变化而变化和调节。在一系列实验中，我们将探索动力蛋白与动力蛋白和动力蛋白-2的相互作用，使用标准生化方法和体外实验相结合，其中从分离的成分中重建双向头和囊泡运动。动力蛋白-马达相互作用的结构基础将通过电子显微镜成像在动力蛋白和动力蛋白或动力蛋白-2之间形成的复合物来确定。这项工作将利用最近在阐明动力蛋白结构方面取得的进展，以及我们小组最近获得的动力蛋白3D重建。研究的第二个方向是动力学蛋白如何与细胞膜相互作用。我们已经确定了一个动力蛋白亚基p27，它似乎在膜结合中起关键作用。P27有许多独特的性质。它存在于动力蛋白分子的一端，与其他动力蛋白亚基不同，它有能力从动力蛋白结构的其余部分释放出来。P27在有丝分裂中也被磷酸化，这表明这是一种以细胞周期依赖的方式控制动力蛋白功能和货物选择的机制。我们的RNAi研究表明p27在有丝分裂中以一种意想不到的方式参与动动蛋白的功能。缺乏p27的细胞在有丝分裂进入的时间和细胞分裂的最后事件中表现出缺陷，但它们的纺锤体是完全正常的，不像动力蛋白功能以其他方式受到干扰时所看到的那样。我们认为p27是在有丝分裂开始和结束时dynactin/motor复合物释放和重新结合到膜上所必需的，并且dynactin募集的这种动态循环对于负责细胞动力学许可的分子机制的运输是必要的。这个新颖的假设将在这里使用活细胞成像和亚细胞分离技术进行测试。

DESCRIPTION (provided by applicant): Cells use microtubule-based motors for a wide range of functions. Some motors are regulated by extrinsic factors that target them to different cargoes or enhance or suppress enzymatic activity. One such factor is dynactin, a highly conserved, multiprotein complex that is ubiquitous among eukaryotes. Dynactin is best known for its contributions to cytoplasmic dynein function, but also appears to work with another motor, kinesin-2. In this proposal, I describe work that addresses two important questions: how dynactin interacts with these two motors to allow coordinated bidirectional movement and how dynactin function changes and is regulated as cells transit the cell cycle. In one series of experiments, we will explore the interactions of dynactin with dynein and kinesin-2, using a combination of standard biochemical methods and in vitro assays in which bidirectional bead and vesicle motility is reconstituted from isolated components. The structural bases of dynactin-motor interactions will be determined using electron microscopy to image complexes formed between dynactin and dynein or kinesin-2. This work will capitalize on recent advances that have been made toward elucidating the structure of dynein, as well as a recent 3D reconstruction of dynactin obtained by our group. A second line of investigation will pursue the question of how dynactin interacts with endomembranes. We have identified one dynactin subunit, p27, that appears to play a key role in membrane binding. p27 has a number of unique properties. It is present at one extreme end of the dynactin molecule where, unlike other dynactin subunits, has the capacity to be released from the rest of the dynactin structure. p27 is also phosphorylated in mitosis, which suggests that this is a mechanism for governing dynactin function and cargo choice in a cell cycle-dependent manner. Our RNAi studies indicate that p27 contributes to dynactin function in an unexpected way in mitosis. Cells lacking p27 show defects in the timing of mitotic entry and the very final events of cytokinesis, but their spindle apparatus is completely normal, unlike what is seen when dynactin function is perturbed in other ways. We believe that p27 is required for release and rebinding of dynactin/motor complexes to membranes at the start and end of mitosis, and that this dynamic cycle of dynactin recruitment is necessary for trafficking of the molecular machinery that is responsible for cytokinetic licensing. This novel hypothesis will be tested here using the techniques of live cell imaging and subcellular fractionation. PUBLIC HEALTH RELEVANCE: Many human pathologies involve perturbations in intracellular transport, subcellular organization and compartment dynamics. The work proposed in this application will help define the basic cellular functions that allow optimal cell health and viability, and will thus provide the foundation for ongoing and future studies of human pathogenesis.