Engineering inhibitable kinesin motors to study axonal transport

设计可抑制的驱动蛋白马达来研究轴突运输

基本信息

批准号：
8427277
负责人：
Kristen J. Verhey
金额：
$ 22.51万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8427277
关键词：
ATP Hydrolysis Acute Address Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animal Model Animals Axon Axonal Transport Behavior Binding Biochemical Biological Biology Carrier Proteins Cell division Cells Charcot-Marie-Tooth Disease Chemicals Communities Complement Cytoplasmic Granules Cytoskeletal Filaments Cytoskeleton Data Defect Development Diagnosis Disease Drosophila genus Dynein ATPase Engineering Ensure Event Family Fibroblasts Filament Funding Mechanisms Future Generations Genes Genetic Engineering Goals Hippocampus (Brain)Human Pathology Huntington Disease In Vitro Intermediate Filaments Intracellular Transport Investigation Kinesin Knowledge Life Link Maintenance Malignant Neoplasms Measures Messenger RNA Methods Microtubules Molecular Motors Motor Motor Activity Movement Mus Mutation Nature Nerve Degeneration Neurites Neurodegenerative Disorders Neurons Organelles Play Process Proteins Public Health Role Shapes Signal Transduction Signaling Molecule Speed Staging Synapses Tertiary Protein Structure Testing Time Transport Process Tubulin Vesicle Work base cell motility cellular imaging chemical genetics ciliopathy fly human disease in vitro Assay inhibitor/antagonist innovation insight interest knock-down multidisciplinary neuron development neuronal cell body novel protein complex prototype research study response small molecule therapeutic target tool trafficking

项目摘要

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.

描述（由申请人提供）：沿着细胞骨架丝驱动货物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，沿着微管丝的基于驱动蛋白的运输在神经元细胞中特别重要。众所周知，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，定义了神经突生长、细胞极化过程中每个驱动蛋白马达的确切运输贡献，由于缺乏以敏锐和运动特异性方式控制运输的方法，轴突的生成和寻路以及电路的形成受到阻碍。在这项提案中，我们将采用“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。使用 kinesin-1 作为原型，我们将使用可被细胞渗透性抑制剂化学靶向的序列对 kinesin-1 的运动结构域进行基因修饰。我们将首先使用体外测定来表征可抑制马达，这些测定产生定量测量运动活动。成功的抑制策略将通过活细胞成像来表征成纤维细胞和初级海马神经元来定义驱动蛋白-1 货物的精确补充。这项工作将为未来的工作奠定基础，目标是：a) 开发用于研究驱动蛋白-1 运输的动物模型；b) 生成驱动蛋白-2 和驱动蛋白-3 家族的可抑制马达。这项工作将为驱动蛋白马达如何引起细胞内蛋白质复合物的协调运输提供令人兴奋的新见解，并将提出人类疾病的治疗靶点。