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

基本信息

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
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/9905964
关键词：
Adaptor Signaling Protein Alzheimer&apos 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

项目摘要

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

联系人 PD/PI：Yildiz、Ahmet 项目概要真核细胞的复杂性需要细胞内的组织、协调和运动。为了克服这些挑战，细胞利用 ATP 驱动的分子马达来运输细胞内的沿着细胞骨架轨道单向的组件促进驱动蛋白和细胞质动力蛋白马达。通过向微管的正负端移动来双向运输各种货物 (MT) 分别存在驱动蛋白的详细机制模型，但动力蛋白的机制和调节。我们发现酿酒酵母动力蛋白通过不协调的步伐在 MT 上行走。它的两个催化结构域及其作用机制与移交有显着不同令人惊讶的是，尽管动力蛋白的结构表征最近取得了进展，但其向 MT 负端移动的强烈方向偏好的分子起源仍不清楚。最近，开发了人动力蛋白重组表达系统，为详细研究打开了大门。令人惊讶的是，首次研究了人类动力蛋白的分子机制。在体外，持续运行并产生比酿酒酵母动力蛋白低得多的力，这与人类动力蛋白在细胞内长距离运输大量细胞内货物的能力。研究表明，当人类动力蛋白形成 2.5 MDa 三元复合物（称为作为 DDB）及其辅因子 dynactin 和货物结合适配器 BicD2 在我们的初步工作中，我们表明了这一点。 dynactin 和 BicD2 也显着增强人类动力蛋白的力量产生，表明 DDB 当连接到相同的货物上时，复合物是一个强大的发动机，并且是驱动蛋白的强大对手。该提案的目标是剖析活性人类动力蛋白复合物的机制并确定如何 dynactin 和 BicD2 调节动力蛋白在双向货物运输过程中与驱动蛋白-1 竞争的能力。我们有三个具体目标，首先，利用蛋白质工程和单分子成像，我们将确定。我们还将解决动力蛋白产生负端定向运动的机械成分。通过冷冻电子显微镜 (cryoEM) 构建“反向”的 MT 结合结构，揭示其次，我们将确定电机的哪些部分负责其方向性。自抑制并表征 dynactin 和 BicD2 如何调节机械化学循环、步进模式第三，我们将在体外重建 MT 上的双向货物运输。利用纯化的人驱动蛋白和DDB复合物揭示“拔河”的机制和调控这些电机之间。我们目标的成功将显着增进对基本机械化学的理解人类动力蛋白并了解它如何实现细胞内货物的逆行运输。项目总结/摘要第 6 页