The Role of Cytoplasmic Dynein in Axonal Transport

描述(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。细胞质动力蛋白是膜细胞器和病毒从轴突末端快速逆行运输到细胞体的马达。动力蛋白还参与沿轴突缓慢运输的微管和神经丝的运输。动力蛋白是一个很大的蛋白质复合体，人们对它的功能知之甚少。这项应用将研究货物结合域中不同亚基亚基在动力蛋白与细胞器、细胞骨架细丝和其他蛋白质结合中的作用。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家族的功能作用上。(1)该家族的两个成员tctex 1和RP3是否与不同的蛋白质相互作用？酵母双杂交和蛋白质结合试验将用于鉴定RP3结合伙伴。(2)这两条轻链是否识别了在细胞中扮演不同角色的动力蛋白群体？每个轻链的突变形式将被用于显性-负性方法，以确定突变轻链的表达是否扰乱了基于动力蛋白的运动性的子集。(3)生化分析和体内共表达方法将被用来检验轻链是动力蛋白货物结合结构域中中间链结合所必需的假设。动力蛋白位于顺行的快、慢轴突运输成分中，两类动力蛋白由不同的中间链组成。第四个目的是研究两种顺行动力蛋白池的作用。表位标记的中间链将被导入培养细胞，并将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。中间链-GFP融合蛋白的活细胞成像将被用来确定这两个动力蛋白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.