Engineering inhibitable kinesin motors to study axonal transport

描述（由申请人提供）：驱动货物沿沿着细胞骨架丝运输的分子马达对于细胞分裂、细胞运动、细胞内运输和纤毛功能等过程至关重要。由于细胞的极化性质以及细胞体和突触区域之间的长距离，基于驱动蛋白的沿沿着微管细丝的运输在神经元细胞中特别重要.已知电机驱动的运输过程中的缺陷会导致神经退行性疾病和其他疾病。然而，在神经突生长、细胞极化、 轴突生成和寻路以及回路形成由于缺乏以急性和运动特异性方式控制运输的方法而受到阻碍。在这项提案中，我们将采取“工程化学遗传方法”来产生可以被小分子抑制的驱动蛋白马达。以驱动蛋白-1为原型，我们将对驱动蛋白-1的运动域进行基因修饰，使其具有可被细胞渗透性抑制剂化学靶向的序列。我们将首先使用体外测定来表征可再生马达， 运动活动成功的抑制策略将通过活细胞成像来表征， 成纤维细胞和原代海马神经元来确定驱动蛋白-1货物的确切补体。这项工作将为未来的工作提供基础，旨在a）开发用于研究驱动蛋白-1转运的动物模型和B）产生驱动蛋白-2和驱动蛋白-3家族的可重复马达。这项工作将为驱动蛋白马达如何引起细胞中蛋白质复合物的协调运输提供令人兴奋的新见解，并将为人类疾病提供治疗靶点。

DESCRIPTION (provided by applicant): Molecular motors that drive cargo transport along cytoskeletal filaments are critical for processes such as cell division, cell motility, intracellulr trafficking and ciliary function. Kinesin- based transport along microtubule filaments is particularly important in neuronal cells due to the polarized nature of the cell and the long distances between the cell body and synaptic regions. Defects in motor-driven transport processes are known to contribute to neurodegenerative and other diseases. Yet defining the exact transport contribution of each kinesin motor during neurite outgrowth, cellular polarization, axon generation and pathfinding, and circuit formation has been hindered by a lack of methods to control transport in an acute and motor-specific manner. In this proposal, we will take an "engineered chemical-genetic approach" to generate kinesin motors that can be inhibited by small molecules. Using kinesin-1 as a prototype, we will genetically modify kinesin-1's motor domain with sequences that can be chemically targeted by cell-permeable inhibitors. We will first characterize the inhibitable motors using in vitro assays that yield quantitative measures of motor activity. Successful inhibitory strategies will then be characterized by live cell imaging in fibroblasts and primary hippocampal neurons to define the exact complement of kinesin-1 cargoes. This work will provide the basis for future work aimed at a) developing animal models for studying kinesin-1 transport and b) generating inhibitable motors of the kinesin-2 and kinesin-3 families. This work will provide exciting new insights into how kinesin motors give rise to coordinated transport of protein complexes in cells and will suggest therapeutic targets in human disease.