Understanding the Mechanism and Regulation of the Human Cytoplasmic Dynein Complex

了解人类细胞质动力蛋白复合物的机制和调节

基本信息

批准号：
9267494
负责人：
Ahmet Yildiz
金额：
$ 29.33万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9267494
关键词：
Adaptor Signaling Protein Address Alzheimer&apos s Disease Award Behavior Binding Biophysics Catalytic Domain Cell physiology Cells Chemicals Complex Cryoelectron Microscopy Development Diffuse Disease Dynein ATPase Elements Engineering Equilibrium Eukaryotic Cell Event Exhibits Filament Funding Future Generations Goals Hand Head Human In Vitro Intracellular Transport Investigation Kinesin Knock-out Lead Learning Locomotion Mechanics Methods Microtubules Minus End of the Microtubule Mitosis Modeling Molecular Molecular Conformation Molecular Motors Motor Movement Mutation Neurobiology Pathogenesis Pattern Play Plus End of the Microtubule Production Property Protein Engineering Proteins Recombinants Regulation Research Resolution Role Running Saccharomyces cerevisiae Schizophrenia Stroke Structure System Testing Time Torque Walking War Work cell motility cofactor dynactin human disease imaging modality inhibitor/antagonist lissencephaly microscopic imaging molecular imaging motor neuron degeneration mutant optical traps polarized cell preference public health relevance reconstitution retrograde transport single molecule success tool

项目摘要

DESCRIPTION (provided by applicant): The complexity of eukaryotic cells requires intracellular organization, coordination, and locomotion. To overcome these challenges, cells utilize ATP-driven molecular motors, which transport intracellular components unidirectionally along cytoskeletal tracks. Kinesin and cytoplasmic dynein motors facilitate bidirectional transport of a variety of cargos by moving towards the plus- and minus-ends of microtubules (MTs), respectively. Detailed mechanistic models exist for kinesin, but the mechanism and regulation of dynein motility are still emerging. We found that S. cerevisiae dynein walks on a MT through uncoordinated stepping of its two catalytic domains and its mechanism of action differs significantly from the coordinated hand-over- hand stepping of kinesin. Surprisingly, despite recent advances in structural characterization of dynein, the molecular origin of its strong directional preference to move towards the MT minus-end remains unclear. Recently, a recombinant expression system was developed for human dynein, opening the doors for detailed studies of its molecular mechanism for the first time. Surprisingly, human dynein exhibited only short possessive runs and produces significantly lower forces than S. cerevisiae dynein in vitro, inconsistent with the ability of human dynein to transport large intracellular cargos over long distances inside cells. New work has revealed that processivity of human dynein is activated when it forms a 2.5 MDa ternary complex (referred to as DDB) with its cofactor dynactin and a cargo binding adaptor BicD2. In our preliminary work, we showed that dynactin and BicD2 also significantly enhance human dynein's force generation, suggesting that the DDB complex is a strong motor and a formidable opponent of kinesin when attached to the same cargo. The goal of this proposal is to dissect the mechanism of active human dynein complexes and determine how dynactin and BicD2 regulate dynein's ability to compete against kinesin-1 during bidirectional cargo transport. We have three specific aims. First, using protein engineering and single-molecule imaging, we will identify the mechanical components of dynein that give rise to its minus-end directed motility. We will also solve the MT-bound structure of "reverse directionality" constructs via cryo-electron microscopy (cryoEM) to reveal the structural basis of dynein directionality. Second, we will identify which part(s) of the motor is responsible for its autoinhibition and characterize how dynactin and BicD2 regulate the mechanochemical cycle, stepping pattern and force generation of human dynein. Third, we will reconstitute bidirectional cargo transport on MTs in vitro using purified human kinesin and DDB complexes and reveal the mechanism and regulation of "tug-of-war" between these motors. Success of our aims will significantly advance the understanding of the fundamental mechanochemistry of human dynein and learn how it achieves retrograde transport of intracellular cargos.

描述（由应用提供）：真核细胞的复杂性需要细胞内组织，协调和运动。为了克服这些挑战，细胞利用ATP驱动的分子电动机，这些电动机在细胞骨骼轨道沿单个方向上的细胞内组件进行运输。动力素和细胞质动力蛋白电动机促进双向转运分别朝着微管（MTS）的加正端和减端移动，各种货物。有详细的机械模型用于驱动蛋白，但是动力蛋白运动的机理和调节仍在出现。我们发现，酿酒酵母动力蛋白在MT上行走，通过其两个催化结构域的不协调阶梯，其作用机理与驱动蛋白的协调手动踩踏显着区别。令人惊讶的是，尽管动力蛋白的结构表征最近取得了进步，但其强烈方向偏好朝着MT减去末端的分子起源尚不清楚。最近，针对人动力蛋白开发了重组表达系统，为首次详细研究其分子机制开了大门。令人惊讶的是，人动力蛋白仅暴露于短势跑，并产生的力明显低于酿酒酵母动力蛋白的体外，这与人动力蛋白在细胞内部长距离内运输大型细胞内cargos的能力不一致。新作品表明，当人类动力蛋白形成2.5 MDA三元复合物（称为DDB）时，其均具有其辅因子dynactin和货物结合适配器BICD2时，它会激活。在我们的初步工作中，我们证明了Dynactin和BICD2也显着增强了人类动力蛋白的产生，这表明DDB复合物是强大的电动机，并且是连接到同一货物时的驱动蛋白的强大选择。该提案的目的是剖析主动人类动力蛋白复合物的机制，并确定Dynactin和BICD2如何调节Dynein在双向货物运输过程中与运动蛋白-1竞争的能力。我们有三个具体的目标。首先，使用蛋白质工程和单分子成像，我们将确定动力蛋白的机械成分，从而导致其负端定向运动。我们还将通过冷冻电子显微镜（Cryoem）解决“反向方向性”构建体的MT结合结构，以揭示动力蛋白方向性的结构基础。其次，我们将确定电动机的自身抑制作用的原因，并表征了Dynactin和BICD2如何调节人体动力蛋白的机械化学循环，步进模式和力产生的力。第三，我们将使用纯化的人类动力蛋白和DDB复合物在体外重新建立双向货物运输，并揭示这些电动机之间“拖船”的机理和调节。我们的目标的成功将大大提高人们对人动力蛋白的基本机制的理解，并了解其如何实现细胞内碳的逆行运输。