The Interaction of Cytoplasmic Dynein and Dynactin

细胞质动力蛋白和动力蛋白的相互作用

• 批准号: 9111914
• 负责人: Erika L Holzbaur
• 金额: $ 36.18万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9111914
• 关键词: Active Biological Transport; Address; Aging; Amyotrophic Lateral Sclerosis; Automobile Driving; Axon; Axonal Transport; Binding; Binding Proteins; Biological Assay; Cell physiology; Cells; Coupled; Cryoelectron Microscopy; Cytoskeleton; Data; Defect; Disease; Distal; Dynein ATPase; Eukaryota; Future; Health; Homeostasis; Image; In Vitro; Kinesin; Length; Life; Lipids; Mediating; Microscopy; Microtubules; Modeling; Molecular; Molecular Motors; Motor; Motor Activity; Movement; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Pathogenesis; Plus End of the Microtubule; Process; Proteins; Recruitment Activity; Regulation; Resolution; Role; Scaffolding Protein; Syndrome; Testing; Transport Vesicles; Vesicle; Vesicle Transport Pathway; Work; biophysical properties; cell motility; dynactin; genetic regulatory protein; improved; in vitro Assay; insight; live cell imaging; meter; motor neuron degeneration; novel; polarized cell; reconstitution; retrograde transport; single molecule; targeted treatment; tool

DESCRIPTION (provided by applicant): Molecular motors drive the active transport of organelles and vesicles along the cellular cytoskeleton. This transport is required for normal cellular function in higher eukaryotes, but is critically important in highly polarized cells such s neurons. Neurons extend axons that can reach up to 1 meter in length. Axons must be continually supplied with proteins and organelles from the cell body~ clearance of aging proteins and dysfunctional organelles are also required to maintain cellular homeostasis. Thus, axonal transport driven by the coordinated activities of cytoplasmic dynein and kinesin motors is essential, and mutations in the motors that drive this transport cause neurodegeneration. Here we focus on understanding the molecular coordination of dynein and kinesin motors driving axonal transport, using the synergistic approaches of live cell imaging and in vitro reconstitution with single molecule resolution to understand the mechanisms involved. We will focus on three specific aims: (1) how does dynactin activate dynein-driven transport? We hypothesize that the binding of dynein to dynactin activates the motor by enhancing recruitment to the microtubule and inducing a shift from diffusive movement to processed motility. Dynactin is a required activator for most dynein-driven functions in the cell. Importantly, however, it is still not clear how dynactin activates dynein. Using newly developed tools and approaches, we will test three specific hypotheses: (i) binding to dynactin enhances recruitment of dynein to the microtubule~ (ii) binding to dynactin enhances dynein's processivity~ and (iii) dynein regulatory proteins dynactin and Lis1 synergistically activate dynein during long-distance axonal transport. (2) What are the mechanisms regulating the engagement of dynein-driven retrograde transport? We hypothesize that there is a spatially-specific mechanism for retrograde transport initiation in the neuron, involving the ordered recruitment of microtubule plus-end binding proteins, dynactin and dynein that is required for normal neuronal function. We will test this hypothesis by addressing the following questions: (i) is binding to CLIP170 necessary and sufficient to explain the recruitment of dynactin to dynamic microtubule plus-ends leading to retrograde transport initiation? And, (ii) what regulates retrograde transport initiation in the neuron? (3) What are he mechanisms coordinating bidirectional organelle transport along the axon? We hypothesize that scaffolding proteins regulate dynein and kinesin motors bound to cargo, and that motors are clustered on the vesicle to facilitate this coordination. We will use live imaging in primary neurons as well as structural approaches including super-resolution microscopy and cryoEM to examine the role of the scaffolding protein JIP1, and the clustering of motors on axonal transport vesicles. Together, these approaches should provide important new information on the mechanisms of motor function during organelle transport. As disruption of these mechanisms leads to neurodegeneration, continued progress will provide new insights into the pathogenesis of neurodegenerative disease and offer new opportunities for targeted therapies.

描述（由申请人提供）：分子电动机沿细胞骨骼驱动细胞器和囊泡的主动转运。这种转运是在较高的真核生物中正常细胞功能所必需的，但在高度极化的细胞中至关重要。神经元延伸轴突，最多可达到1米长。轴突必须不断提供细胞体的蛋白质和细胞器〜衰老蛋白质和功能失调细胞器的清除率也需要维持细胞稳态。因此，由细胞质动力蛋白和驱动蛋白电动机的协调活性驱动的轴突转运至关重要，并且在驱动该转运的电动机中的突变会导致神经变性。在这里，我们专注于使用活细胞成像的协同方法和体外重新建立的协同方法来理解动力蛋白和驱动蛋白电动机的分子协调 通过单分子分辨率了解所涉及的机制。我们将重点关注三个特定目标：（1）Dynactin如何激活动力蛋白驱动的运输？我们假设动力蛋白与dynactin的结合通过增强对微管的募集并诱导从扩散运动到加工运动的转移来激活电动机。 Dynactin是细胞中大多数动力蛋白驱动功能的必需激活剂。但是，重要的是，仍然不清楚 Dynactin如何激活动力蛋白。 Using newly developed tools and approaches, we will test three specific hypotheses: (i) binding to dynactin enhances recruitment of dynein to the microtubule~ (ii) binding to dynactin enhances dynein's processivity~ and (iii) dynein regulatory proteins dynactin and Lis1 synergistically activate dynein during long-distance axonal transport. （2）调节动力蛋白驱动的逆行运输的机制是什么？我们假设存在一种在神经元中逆行转运启动的空间特异性机制，涉及正常神经元功能所需的微管加末端结合蛋白，dynactin和dynein的有序募集。我们将通过解决以下问题来检验这一假设：（i）与clip170结合，足以解释dynactin募集到动态微管加末端，从而导致逆行运输启动？ （ii）是什么调节神经元中的逆行运输启动？ （3）他在轴突沿着双向细胞器转运的协调的机制是什么？ 我们假设脚手架蛋白调节与货物结合的动力蛋白和驱动蛋白电动机，并将电动机聚集在囊泡上以促进这种配位。我们将在原发性神经元中使用实时成像以及包括超分辨率显微镜和冷冻的结构方法来检查脚手架蛋白JIP1的作用，以及电动机在轴突传输囊泡上的聚类。总之，这些方法应提供有关细胞器传输过程中运动功能机制的重要新信息。随着这些机制的破坏导致神经变性，持续的进步将为神经退行性疾病的发病机理提供新的见解，并为有针对性的疗法提供新的机会。