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

• 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
• 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' 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

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(申请人提供)：驱动沿细胞骨架细丝运输货物的分子马达对细胞分裂、细胞运动、细胞内运输和纤毛功能等过程至关重要。由于神经元细胞的极化性质以及细胞体和突触区域之间的较长距离，基于动蛋白的沿微管细丝的运输在神经细胞中尤为重要。众所周知，运动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。但确定了在轴突生长过程中每个运动蛋白马达的确切运输贡献，细胞极化， 由于缺乏以急性和运动特异性方式控制运输的方法，轴突的产生和寻径以及回路的形成一直受到阻碍。在这项提议中，我们将采用一种“化学-遗传工程方法”来产生可以被小分子抑制的动蛋白马达。以Kinesin-1为原型，我们将用细胞通透性抑制剂可以化学靶向的序列对Kinesin-1‘S运动区进行基因修饰。我们将首先使用体外测试来表征可抑制的发动机，这些测试产生了定量的测量 运动活动。成功的抑制策略将通过活细胞成像来表征 成纤维细胞和原代海马神经元，以确定Kinesin-1货物的确切补充。这项工作将为未来的工作提供基础，旨在a)建立研究kinesin-1转运的动物模型，以及b)产生kinesin-2和kinesin-3家族的可抑制马达。这项工作将为运动蛋白马达如何在细胞内引起蛋白质复合体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。 公共卫生相关性：这项提案中的工作与公共健康相关，因为许多人类疾病，如神经退行性变、癌症和纤毛病，都是由运动蛋白在运输事件中的失调引起的。这项拟议的研究将确定和表征运动蛋白的关键行为，并将促进我们对运动运输的理解，以促进运动相关疾病的治疗。