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 真核细胞的复杂性要求细胞内的组织、协调和运动。至 克服这些挑战，细胞利用ATP驱动的分子马达，在细胞内运输 组件沿细胞骨架轨道单向排列。动蛋白和细胞质动力蛋白马达促进 通过向微管正负两端移动实现各种货物的双向运输 (MTS)。目前已有关于动蛋白的详细机制模型，但动力蛋白的机制和调节 动力仍在不断涌现。我们发现，酿酒酵母动力蛋白通过不协调的步态在MT上行走 它的两个催化结构域及其作用机制与配位移交显著不同。 动蛋白的手步。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 其强烈的定向偏好朝向MT负端移动的分子起源尚不清楚。 最近，一种人动力蛋白的重组表达系统被开发出来，为详细的 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很短的时间 在体外，进程运行并产生的力量明显低于酿酒酵母动力蛋白，这与 人类动力蛋白在细胞内远距离运输大型细胞内货物的能力。新作品有了 揭示了当人动力蛋白形成2.5-丙二醛三元络合物时，它的加工性被激活。 如DDB)及其辅因子动力蛋白和货物结合接头BicD2。在我们的初步工作中，我们表明 动力蛋白和BicD2也显著增强了人类动力蛋白的力量生成，这表明DDB Complex是一种强大的发动机，当连接到相同的货物上时，它是Kinesin的强大对手。 这项提议的目标是剖析活性人类动力蛋白复合体的机制，并确定如何 动力蛋白和BicD2调节动力蛋白在双向货物运输过程中与动蛋白-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，我们将识别 动力蛋白的机械成分，导致其负端定向运动。我们还将解决 用低温电子显微镜(CryoEM)研究“反向”构造物的MT结合结构 动力蛋白方向性的结构基础。其次，我们将确定电机的哪个部分(S)负责其 自我抑制和表征dynactin和BicD2如何调节机械力化学循环、步进模式 以及人类动力蛋白的力量产生。第三，我们将在体外重建MTS上的双向货物运输 利用纯化的人激动素和DDB复合体揭示拔河的机制和调节 在这些马达之间。 我们的目标的成功将极大地促进对基本机械力化学的理解 并学习它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