The Role of Cytoplasmic Dynein in Axonal Transport

描述（由申请人提供）：神经元需要轴突运输来建立和维持轴突和突触。细胞质动力蛋白是将膜性细胞器和病毒从轴突末端快速逆行运输到细胞体的马达。动力蛋白还参与微管和神经丝沿着轴突的缓慢运输。动力蛋白是一种大的蛋白质复合物，人们对它的功能知之甚少。本申请将研究不同亚型的亚基的货物结合域中的动力蛋白结合细胞器，细胞骨架丝，和其他蛋白质的作用。最近的证据表明，动力蛋白轻链对于动力蛋白与特定蛋白质的结合是重要的。前三个目标集中在tctex 1家族的功能作用。(1)家族的两个成员tctex 1和rp 3与不同的蛋白质相互作用吗？酵母双杂交和蛋白结合试验将用于鉴定rp 3结合伴侣。(2)这两条轻链能识别细胞中不同作用的动力蛋白群吗？将在显性阴性方法中使用每条轻链的突变形式，以确定突变轻链的表达是否破坏基于动力蛋白的运动性的子集。(3)将使用生化分析和体内共表达方法来检验轻链是动力蛋白货物结合结构域中中间链缔合所必需的假设。动力蛋白是轴突顺行快、慢运输的两种组分，两种动力蛋白由不同的中间链组成。第四个目的是研究两种动力蛋白顺行池的作用。将表位标记的中间链转染到培养的细胞中，并使用免疫细胞化学来确定具有不同中间链的动力蛋白是否与不同的细胞器或细胞骨架结构结合。将使用中间链-GFP融合蛋白的活细胞成像来确定动力蛋白的两个池是否具有不同的顺行和逆行运动特性。这些研究将为轴突运输机制提供有价值的信息，轴突运输是轴突生长和再生的重要神经元过程。

DESCRIPTION (provided by applicant: Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for fast retrograde transport of membranous organelles and viruses from the axon terminus to the cell body. Dynein is also implicated in the transport of microtubules and neurofilaments along the axon in slow transport. Dynein is a large protein complex and very little is known about how it functions. This application will investigate the role of different isoforms of subunits in the cargo-binding domain in dynein binding to organelles, cytoskeletal filaments, and other proteins. Recent evidence suggests that the dynein light chains are important for dynein binding to specific proteins.The first three aims focus on the functional role of the tctex1 family. (1) Do the two members of the family, tctex1 and rp3, interact with different proteins? Yeast two-hybrid and protein binding assays will be used to identify rp3 binding partners. (2) Do the two light chains identify dynein populations with different roles in cells? Mutated forms of each of the light chains will be used in a dominant-negative approach to determine if expression of a mutated light chain disrupts a subset of dynein-based motility. (3) Biochemical analyses and in vivo co-expression methods will be used to test the hypothesis that the light chains are necessary for the association of intermediate chains in the dynein cargo-binding domain. Dynein is in the anterograde fast and slow axonal transport components and the two populations of dynein are composed of different intermediate chains.The fourth aim focuses on the roles of the two anterograde pools of dynein. Epitope-tagged intermediate chains will be transfected into cultured cells and immunocytochemistry will be used to determine if dyneins with different intermediate chains are bound to different organelles or cytoskeletal structures. Live cell imaging of intermediate chain-GFP fusion proteins will be used to determine if the two pools of dynein have different anterograde and retrograde motility properties. These studies will contribute valuable information about the mechanism of axonal transport, a neuronal process important for axon growth and regeneration.