Molecular mechanism of bidirectional transport

项目摘要 细胞内囊泡和细胞器的双向运输涉及微管马达之间的拔河 驱动蛋白和动力蛋白。这种转运在神经元的轴突和树突以及纤毛和神经纤维中特别重要。 鞭毛和运输缺陷也与阿尔茨海默氏症和ALS等神经退行性疾病有关 纤毛病变虽然许多分子的球员是已知的，这些组件的工作机制， 以及它们的活动如何联合收割机来实现双向运输的涌现属性， 充分理解。这项建议的目的是弥合 单个驱动蛋白和动力蛋白马达的机械化学和囊泡的双向运输动力学， 在细胞中观察到的细胞器。未解决的问题包括：负载如何影响机械化学， 不同驱动蛋白和动力蛋白的分离动力学？相反的马达如何协调和竞争， 实现双向运输？调控蛋白、微管相关蛋白和微管蛋白是如何在细胞凋亡后， 翻译修饰改变了正末端和负末端定向运动的平衡，以实现适当的载体功能。 交通工具？为了解决这些问题，干涉散射（iSCAT）显微镜与纳米空间 精确度和毫秒级时间分辨率将用于跟踪单个运动域、单个运动 蛋白质和多马达组装，因为它们沿着它们的微管轨道。这些显微镜研究将 通过停流动力学研究、体外重建实验和计算 建模以了解日益复杂的装配。有待研究的具体运动机制 包括驱动蛋白-3的快速和超持续合成能力的起源，驱动蛋白-5的聚合酶机制， 以及动力蛋白被其衔接蛋白激活的分子基础。将研制一种DNA张力计， 了解机械负荷对驱动蛋白和动力蛋白机械化学的影响，统计工具将 开发用于从这些实验中提取负载依赖的脱离动力学。最后，多电机 组装将使用DNA折纸，这允许精确控制电机数量和定位， 和重构的脂质囊泡，其模拟细胞内货物的机械和扩散性质。这 这项工作将推进我们对细胞器如何在细胞中正确定位以及细胞器如何特异性地定位的理解。 细胞内货物被可靠地靶向其适当的细胞位置。

PROJECT SUMMARY Bidirectional transport of vesicles and organelles in cells involves a tug-of-war between the microtubule motors kinesin and dynein. This transport is particularly important in the axons and dendrites of neurons and in cilia and flagella, and transport defects are linked to neurodegenerative diseases such as Alzheimer’s and ALS, as well as ciliopathies. Although many of the molecular players are known, the working mechanisms of these component parts and how their activities combine to achieve the emergent property of bidirectional transport are not sufficiently understood. The goal of this proposal is to bridge the gulf in understanding between the mechanochemistry of single kinesin and dynein motors and the bidirectional transport dynamics of vesicles and organelles observed in cells. Unresolved questions include: How does load affect the mechanochemistry and detachment kinetics of different kinesins and dynein? How do opposing motors coordinate and compete to achieve bidirectional transport? How do regulatory proteins, microtubule associated proteins and tubulin post- translational modifications alter the balance of plus- and minus-end directed motility to achieve proper vectorial transport? To address these questions, Interferometric Scattering (iSCAT) microscopy with nanometer spatial precision and millisecond temporal resolution will be used to track individual motor domains, single motor proteins, and multi-motor assemblies as they step along their microtubule tracks. These microscopy studies will be complemented by stopped-flow kinetics investigations, in vitro reconstitution experiments, and computational modeling to understand assemblies of increasing complexity. Specific motor mechanisms to be investigated include the origin of the fast speed and superprocessivity of kinesin-3, the polymerase mechanism of kinesin-5, and the molecular basis of dynein activation by its adapter proteins. A DNA tensiometer will be developed to understand the influence of mechanical load on kinesin and dynein mechanochemistry, and statistical tools will be developed to extract load-dependent detachment kinetics from these experiments. Finally, multi-motor assemblies will be built using DNA origami, which allows for precise control of motor number and positioning, and reconstituted lipid vesicles, which mimic the mechanical and diffusional properties of intracellular cargo. This work will advance our understanding of how organelles are correctly positioned in cells and how specific intracellular cargo are reliably targeted to their proper cellular locations.