Regulation of Cytoplasmic Dynein Based Vesicle Transport

描述（由申请人提供）：细胞使用基于微管的马达来实现广泛的功能。一些马达受外在因素调节，这些外在因素将它们靶向不同的货物或增强或抑制酶活性。其中一个因子是dynactin，一种高度保守的多蛋白复合物，在真核生物中普遍存在。Dynactin最为人所知的是其对细胞质动力蛋白功能的贡献，但似乎也与另一种马达驱动蛋白-2一起工作。在这个建议中，我描述的工作，解决了两个重要的问题：如何dynactin与这两个电机相互作用，以允许协调的双向运动，以及dynactin功能如何变化，并作为细胞转运细胞周期的调节。在一系列的实验中，我们将探讨dynactin与动力蛋白和驱动蛋白-2的相互作用，使用标准的生物化学方法和体外试验的组合，其中双向珠和囊泡的运动是从分离的组件重建。动力蛋白-运动相互作用的结构基础将使用电子显微镜来确定，以成像动力蛋白和动力蛋白或驱动蛋白-2之间形成的复合物。这项工作将利用最近的进展，已经朝着阐明动力蛋白的结构，以及最近的三维重建动力蛋白获得了我们的小组。第二条研究路线将探讨dynactin如何与内膜相互作用的问题。我们已经确定了一个dynactin亚基，p27，似乎在膜结合中发挥了关键作用。p27具有许多独特的性质。它存在于动力肌动蛋白分子的一端，与其他动力肌动蛋白亚基不同，它能够从动力肌动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，这表明这是以细胞周期依赖性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有助于dynactin功能。缺乏p27的细胞在有丝分裂进入的时间和胞质分裂的最后事件上表现出缺陷，但它们的纺锤体是完全正常的，不像当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是需要释放和重新绑定的dynactin/电机复合物的膜在有丝分裂的开始和结束，而这种动态循环的dynactin招聘是必要的贩运的分子机制，负责细胞动力学许可。这一新的假设将在这里使用活细胞成像和亚细胞分级分离技术进行测试。 公共卫生相关性：许多人类病理学涉及细胞内转运、亚细胞组织和区室动力学的扰动。本申请中提出的工作将有助于定义允许最佳细胞健康和活力的基本细胞功能，从而为正在进行和未来的人类发病机制研究提供基础。

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.