Understanding the Mechanism and Regulation of the Human Cytoplasmic Dynein Complex

 描述(申请人提供)：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，沿着细胞骨架轨迹单向运输细胞内的成分。动蛋白和胞浆动力蛋白马达促进双向转运 通过分别向微管(MT)的正端和负端移动，对各种货物进行检测。目前已有关于动力蛋白的详细机制模型，但动力蛋白运动的机制和调节仍在不断涌现。我们发现，酿酒酵母dynein通过其两个催化域的非协调步进在MT上行走，其作用机制与Kinesin的协调手步行走有显着不同。令人惊讶的是，尽管动力蛋白最近在结构表征方面取得了进展，但其强烈的定向偏好朝向MT-末端移动的分子来源仍不清楚。最近，人类动力蛋白的重组表达系统被开发出来，首次为详细研究其分子机制打开了大门。令人惊讶的是，人动力蛋白在体外只表现出短暂的占有性，并且产生的力量明显低于酿酒酵母动力蛋白，这与人动力蛋白在细胞内长距离运输大型细胞内货物的能力不一致。新的研究表明，当人类动力蛋白与其辅因子动力蛋白和货物结合接头BicD2形成2.5MDA三元复合体(称为DDB)时，其加工性被激活。在我们的初步工作中，我们发现dynactin和BicD2也显著增强了人类dynein的力量生成，这表明当DDB复合体附着在相同的货物上时，它是一个强大的发动机和kinesin的强大对手。这项建议的目的是剖析活性的人类动力蛋白复合体的机制，并确定在双向货物运输过程中，动力蛋白和BicD2如何调节动力蛋白与激动素-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，我们将确定动力蛋白的机械成分，导致其负端定向运动。我们还将通过冷冻电子显微镜(Cryo-EM)解决“逆方向性”结构的MT结合结构，以揭示动力蛋白方向性的结构基础。其次，我们将确定发动机的哪个部分(S)负责其自身抑制，并表征动力蛋白和BicD2如何调节人类动力蛋白的机械力化学循环、步进模式和力的产生。第三，我们将利用纯化的人激动素和DDB复合体在体外重建MTS上的双向货物运输，并揭示这些马达之间的拉锯战机制和调节。我们的目标的成功将极大地促进对人类动力蛋白基本机械力化学的理解，并了解它如何实现细胞内货物的逆行运输。

 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.