A Request for a Fluorescence Microscope Coupled with CLiC Technology to Image Single Molecules at High Concentrations in Real Time

要求荧光显微镜与 CLiC 技术相结合，对高浓度单分子进行实时成像

• 批准号: 9905964
• 负责人: Ahmet Yildiz
• 金额: $ 2.3万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 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/9905964
• 关键词: Adaptor Signaling Protein; Alzheimer' s Disease; Binding; Catalytic Domain; Cells; Chemicals; Complex; Coupled; Cryoelectron Microscopy; Development; Disease; Dynein ATPase; Eukaryotic Cell; Exhibits; Future; Generations; Goals; Hand; Human; Image; In Vitro; Intracellular Transport; Investigation; Kinesin; Knock-out; Lead; Learning; Locomotion; Mechanics; Microtubules; Minus End of the Microtubule; Mitosis; Modeling; Molecular; Molecular Motors; Motor; Movement; Mutation; Neurobiology; Pathogenesis; Pattern; Protein Engineering; Recombinants; Regulation; Role; Running; Saccharomyces cerevisiae; Schizophrenia; Structure; System; Technology; Time; Walking; War; Work; cell motility; cofactor; dynactin; fluorescence microscope; human disease; inhibitor/antagonist; lissencephaly; molecular imaging; motor neuron degeneration; mutant; preference; reconstitution; retrograde transport; single molecule; success

Contact PD/PI: Yildiz, Ahmet Project Summary 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 processive 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. Project Summary/Abstract Page 6

联系PD/PI：Yildiz，Ahmet 项目摘要 真核细胞的复杂性需要细胞内组织，协调和运动。到 克服这些挑战，细胞利用ATP驱动的分子电动机，这些电动机转运细胞内 沿细胞骨架轨道单向分量。制备的动力素和细胞质动力蛋白电动机 通过朝着微管的正端和减端移动，将各种货物的双向运输 （MT）分别。有详细的机械模型，但动力蛋白的机理和调节 运动仍在出现。我们发现，酿酒酵母动力蛋白通过不协调的踏脚行走 在其两个催化结构域中，其作用机理在 手动踩踏器。令人惊讶的是，Dospite在结构表征的最新进展，Dynein， 其强烈方向偏好向MT减去末端移动的分子起源尚不清楚。 最近，为人动力蛋白开发了重组表达系统，打开了大门以获取细节 首次研究其分​​子机制。令人惊讶的是，人类动力蛋白只表现出简短 在体外，过程运行和产生的力明显低于酿酒酵母动力蛋白，与 人动力蛋白在细胞内部长距离内输送大细胞内肉类的能力。新作品有 揭示了人动力蛋白的加工性在形成2.5 MDA三元复合物时被激活（称为 作为DDB）及其辅因子大氨酸和一个货物结合适配器BICD2。在我们的初步工作中，我们表明 Dynactin和BICD2也显着增强了人动力蛋白的产生，表明DDB 复合物是连接到同一货物时的强力电动机，也是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，并确定如何 Dynactin和BICD2调节Dynein在双向货物运输过程中与动力蛋白-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产生其负端的定向运动。我们还将解决 通过冷冻电子显微镜（Cryoem）构建的“反向定向”构建的MT结构结构，以揭示 动力蛋白方向的结构基础。其次，我们将确定电动机的哪一部分负责 自身抑制并表征了Dynactin和BICD2如何调节机械化学周期，步进模式 并产生人类动力蛋白。第三，我们将在体外重新建立双向货物运输 使用纯化的人类动力蛋白和DDB复合物，并揭示“拔河战”的机制和调节 在这些电动机之间。 我们的目标的成功将大大提高人们对的基本机械化学的理解 人动力蛋白，学习如何实现细胞内碳的逆行运输。 项目摘要/摘要页面6