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

联系PD/PI：Yildiz，Ahmet 项目摘要 真核细胞的复杂性需要细胞内的组织、协调和运动。到 为了克服这些挑战，细胞利用ATP驱动的分子马达， 成分单向沿着细胞骨架轨道。驱动蛋白和细胞质动力蛋白马达促进 通过向微管的正端和负端移动来双向运输各种货物 (MTs)，分别。驱动蛋白存在详细的机制模型，但动力蛋白的机制和调节 运动性仍在出现。我们发现S.酿酒动力蛋白通过不协调的步进在MT上行走 它的两个催化域及其作用机制与协调的移交有很大不同， 驱动蛋白的手动步进。令人惊讶的是，尽管最近在动力蛋白的结构表征方面取得了进展， 其向MT负端移动的强烈定向偏好的分子起源仍不清楚。 最近，开发了人动力蛋白的重组表达系统，为详细研究动力蛋白打开了大门。 首次对其分子机制进行了研究。令人惊讶的是，人类动力蛋白只表现出短的 进行性运动产生的力明显低于S。酿酒酵母动力蛋白体外，不符合 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。新作 揭示了人动力蛋白的持续合成能力在其形成2.5MDa三元复合物时被激活（参见 作为DDB）及其辅因子动力蛋白和货物结合衔接子BicD 2。在我们的初步工作中，我们表明， dynactin和BicD 2也显著增强了人类动力蛋白的力产生，这表明DDB 复合体是一个强大的马达，当附着在同一货物上时，它是驱动蛋白的强大对手。 这项提案的目标是剖析活跃的人类动力蛋白复合体的机制，并确定如何 动力蛋白和BicD 2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，使用蛋白质工程和单分子成像，我们将识别 动力蛋白中产生负端定向运动的机械成分。我们还将解决 通过冷冻电子显微镜（cryoEM）观察“反向”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，我们将确定电机的哪些部分负责其 自抑制，并表征dynactin和BicD 2如何调节机械化学循环，步进模式 以及人类动力蛋白的产生。第三，我们将在体外重建MT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 在这些发动机之间。 我们的目标的成功将大大推进对基本机械化学的理解， 并了解其如何实现细胞内货物的逆行运输。 项目摘要/摘要第6页

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