How Molecular Motors Work Together to Move Cargo: Nanometer Distances and Piconewton Forces

分子马达如何协同工作来移动货物：纳米距离和皮牛顿力

• 批准号: 10377346
• 负责人: PAUL R SELVIN
• 金额: $ 30.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377346
• 关键词: Affect; Affinity; Alzheimer' s Disease; Automobile Driving; Back; Behavior; Binding; Biological Assay; Bombyx; Buffers; Bypass; Cells; Communication; Coupled; Crowding; DNA; Data; Diffuse; Disease; Dynein ATPase; Environment; Equilibrium; Eukaryotic Cell; Face; Family; Filament; Fluorescence; Future; Human; In Vitro; Individual; Intermediate Filaments; Kinesin; Label; Letters; Measures; Microfilaments; Microtubule-Associated Proteins; Microtubules; Modeling; Modification; Molecular Motors; Motion; Motor; Movement; Mutation; Nerve Degeneration; Parkinson Disease; Peptides; Polymers; Positioning Attribute; Property; Protocols documentation; Quantum Dots; Regulation; Reporting; Resistance; Salts; Series; Sodium Chloride; Speed; Spiders; Streptavidin; Techniques; Testing; Time; Travel; Tubulin; Work; Yeasts; base; density; experimental study; improved; in vitro Assay; in vivo; innovation; insight; nanometer; nanometer resolution; public health relevance; recruit; sensor; single molecule

Abstract Significance: How cytoplasmic cargoes move within a crowded cell, over long distances and speeds that are nearly the same as when moving in a simple buffer, has long been mysterious. Roadblocks, detours and the dense environment apparently do not, on average, slow the cargoes as they move around the cell. Here we report a simple mechanism, based on a new type of in vitro force-gliding assay, where multiple motors operate simultaneously on a common cargo and their forces combine. The cargo is a microtubule that is transported above a series of randomly placed, but far-apart motors that are fixed to a coverslip through a spring. The cargo’s position and velocity are measured via fluorescence; the force of each motor is measured with piconewton accuracy over many minutes by measuring the displacement from equilibrium. For the first time, we have managed to develop an assay to quantitatively measure multiple motors, as opposed to single motors. Tension is the key to communication. One motor creates tension on the microtubule filament that is felt by other motors on the same microtubule. When the microtubule faces an obstacle, the tension increases and more motors get activated to bypass the roadblock. Alternatively, the motor facing the highest resistance lets go, allowing the microtubule to locally diffuse and take a new path. A sharing of force between the motors is critical. The idea is that multiple motors allow the cargo’s speed to be roughly constant in the absence or presence of roadblocks and detours; however, with these impediments, the forces of multiple motors add together, allowing the cargo to smoothly travel through—or around—the obstacles. Examples of roadblocks/detours include different microtubule-associated proteins (MAPs) that can come on and off, as well as actin and intermediate filaments. We use multiple mammalian kinesins or multiple yeast dyneins, and in the future, we will employ multiple kinesins and multiple human cytoplasmic dyneins, the latter experiment asking the question of how, or if, these two opposite-directed families of motors compete or cooperate with each other. We have preliminary data for multiple kinesins and find that they are good sharers and dynamically come on and off the microtubule. We also have data for multiple yeast dyneins, as well as for kinesin and yeast dynein. We find that the molecular motors vary between “hindering” and “driving” positions, which dynamically change as a function of roadblocks. We will also simulate in vivo settings, where, for example, salt concentration and competing filaments are high, or the availability of free motors is limited. The technique presented here is also innovative. Our technique will involve measuring single molecule fluorescence with nanometer resolution; it will involve measuring forces in the piconewton range based on fluorescence using a unique worm-like-chain of either DNA or Polyethyleneglycol with a spider silkworm tension sensor; it will involve measuring multiple motors that all act individually yet work on a single cargo.

抽象的 意义：细胞质货物如何在拥挤的电池内移动，远距离和速度是 与简单的缓冲区移动几乎相同，长期以来一直是神秘的。障碍，弯路和 茂密的环境显然平均不会在货物周围移动时减慢货物的速度。我们在这里 报告一种基于新型的体外力 - 运动测定的简单机制，其中多个电动机运行 同样，在普通货物上及其力量结合在一起。货物是运输的微管 在一系列随机放置但遥远的电动机上方，这些电动机固定在弹簧上的盖玻片上。货物的 位置和速度通过荧光测量；用piconnewton测量每个电动机的力 通过测量平衡的位移，可以在数分钟内进行精确度。我们第一次有 与单电机相比，设法开发了一种定量测量多个电动机的测定法。 紧张是交流的关键。一辆电动机会在微管丝上产生张力 同一微管上的其他电动机。当微管面对障碍时，张力会增加 电动机被激活以绕过障碍。或者，面向最高阻力的电动机放开， 允许微管局部扩散并采用新路径。电动机之间的力共享至关重要。 想法是，多个电动机允许在不存在或存在的情况下，货物的速度大致恒定 障碍和弯路；但是，由于这些障碍，多个电动机的力加在一起，允许 货物可以平稳地穿越或周围的障碍物。障碍/弯路的例子包括 可以打开和关闭的不同微管相关蛋白（地图）以及肌动蛋白和中间体 细丝。 我们使用多种哺乳动物驱素或多种酵母动蛋白，将来我们将采用多个 驱动蛋白和多个人类细胞质动力蛋白，后一个实验询问了一个问题，或者是否是 两个相反的电动机家庭互相竞争或合作。我们有针对的初步数据 多个驱动剂，发现它们是良好的股份，并动态地启动了微管。我们也是 具有多种酵母动蛋白以及驱动蛋白和酵母动蛋白的数据。我们发现分子电动机 在“阻碍”和“驾驶”位置之间有所不同，这些位置随着障碍的函数而动态变化。我们将 还模拟体内设置，例如，盐浓度和竞争丝很高，或者 免费电动机的可用性是有限的。 这里提出的技术也是创新的。我们的技术将涉及测量单分子 分辨率分辨率的荧光；它将涉及基于Piconnewton范围内的测量力 使用蜘蛛蚕张力的DNA或聚乙烯的独特的DNA或聚乙烯乙二醇的荧光 传感器;它将涉及测量所有电动机，这些电动机都可以单独行动但在单个货物上工作。