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：
8292689
负责人：
Kristen J. Verhey
金额：
$ 19.44万
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 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292689
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 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. PUBLIC HEALTH RELEVANCE: The work in this proposal is relevant to public health because many human pathologies, such as neurodegeneration, cancer and the ciliopathies are caused by the disregulation of kinesin transport events. The proposed study will identify and characterize key behaviors of kinesin motor proteins and will advance our understanding of motor transport in order to promote treatment of motor-based diseases.

描述（由申请人提供）：驱动沿细胞骨骼细丝的货物传输的分子电动机对于诸如细胞分裂，细胞运动，ellacellulrrrrr贩运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，在神经元细胞中沿微管丝沿微管丝的转运尤其重要。已知运动驱动的运输过程中的缺陷会导致神经退行性和其他疾病。然而，在神经突产物生长过程中，定义每个驱动蛋白运动的确切转运贡献，细胞极化，缺乏以急性和运动特异性控制运输的方法，阻碍了轴突的产生和探路和电路形成。在此提案中，我们将采用一种“工程化学遗传学方法”来产生可受小分子可以抑制的驱动蛋白电动机。使用kinein-1作为原型，我们将用序列可以通过序列来修改kinesin-1的运动结构域，这些序列可以通过可通过可渗透的抑制剂化学靶向。我们将首先使用体外测定法表征可约束的电动机，以产生定量测量运动活动。然后，成功的抑制策略将以活细胞成像为特征成纤维细胞和原发性海马神经元，以定义驱动蛋白-1货物的精确补体。这项工作将为未来的工作提供旨在a）开发动物模型的未来工作的基础，以研究驱动蛋白-1运输和b）生成可抑制的驱动蛋白-2和驱动蛋白-3家族的电动机。这项工作将为细胞中蛋白质复合物在细胞中的协调运输如何产生蛋白质复合物的协调型，并提出人类疾病中的治疗靶标。公共卫生相关性：该提案中的工作与公共卫生有关，因为许多人类病理（例如神经变性，癌症和纤毛病）是由于疏离了动力素运输事件而引起的。拟议的研究将确定并表征动力蛋白运动蛋白的关键行为，并将促进我们对运动运输的理解，以促进对运动疾病的治疗。