Molecular mechanism of bidirectional transport

双向运输的分子机制

• 批准号: 10353437
• 负责人: William Olaf Hancock
• 金额: $ 81.46万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10353437
• 关键词: Adaptor Signaling Protein; Address; Affect; Alzheimer' s Disease; Axon; Cells; Cilia; Complement; Computer Models; DNA; Defect; Dendrites; Diffusion; Dynein ATPase; Equilibrium; Flagella; Goals; Huntington Disease; In Vitro; Individual; Intracellular Transport; Investigation; Kinesin; Kinetics; Link; Lipids; Location; Mechanics; Microscopy; Microtubule-Associated Proteins; Microtubules; Molecular; Motor; Neurodegenerative Disorders; Neurons; Organelles; Polymerase; Positioning Attribute; Post-Translational Protein Processing; Property; Proteins; Speed; Tubulin; Vesicle; War; Work; cell motility; ciliopathy; experimental study; genetic regulatory protein; mechanical load; millisecond; motor control; nanometer; reconstitution; temporal measurement; tool; vector; vesicle transport

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.

项目总结 细胞内囊泡和细胞器的双向运输涉及微管马达之间的拉锯战 激动素和动力蛋白。这种运输在神经元的轴突和树突以及纤毛和 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 作为纤毛病。虽然许多分子参与者是已知的，但这些组分的工作机制 部分以及它们的活动如何结合以实现双向运输的紧急特性 充分理解。这项提议的目标是弥合两国在理解上的鸿沟 单个动蛋白和动力蛋白马达的机械力化学和小泡和动力蛋白的双向运输动力学 细胞内观察到的细胞器。悬而未决的问题包括：载荷如何影响机械力化学和 不同动蛋白和动力蛋白的脱附动力学？对立的马达如何协调和竞争 实现双向运输？调节蛋白、微管相关蛋白和微管蛋白是如何在 翻译修饰改变了正端和负端定向运动的平衡，以实现适当的向量 交通工具？为了解决这些问题，具有纳米空间的干涉散射(ISCAT)显微镜 精度和毫秒级的时间分辨率将用于跟踪单个马达领域、单个马达 蛋白质，以及沿着微管轨迹行走的多马达组件。这些显微镜研究将 辅以停流动力学研究、体外重建实验和计算 建模以了解日益复杂的部件。具体的运动机制有待研究 包括Kinesin-3快速和超加工的起源，Kinesin-5的聚合机制， 以及动力蛋白被其接头蛋白激活的分子基础。一种DNA张力计将被开发出来 了解机械负荷对动蛋白和动力蛋白机械力化学的影响，统计工具将 被开发来从这些实验中提取依赖于载荷的脱离动力学。最后，多电机 组装将使用DNA折纸技术制造，它允许精确控制电机数量和定位， 和重组脂泡，模拟细胞内货物的机械和扩散特性。这 这项工作将促进我们对细胞器在细胞中如何正确定位以及具体程度的理解 细胞内的货物可以可靠地定位到它们适当的蜂窝位置。