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: 9905534
• 负责人: PAUL R SELVIN
• 金额: $ 29.09万
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905534
• 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细胞内长距离、高速度地移动 几乎就像在简单的缓冲区中移动时一样，早已是神秘莫测的路障、弯路了。 平均而言，密集的环境显然不会减慢货物在细胞内移动的速度。 报告了一种基于新型体外力滑动测定的简单机制，其中多个电机运行 同时作用在一个普通的货物上，它们的力结合在一起，该货物是一个正在运输的微管。 上面是一系列随机放置但相距较远的电机，这些电机通过弹簧固定在盖玻片上。 通过荧光测量位置和速度；每个电机的力通过皮牛顿测量 通过测量平衡位移，我们第一次获得了多分钟的精度。 成功开发了一种定量测量多个电机（而不是单个电机）的方法。 张力是沟通的关键。一个马达在微管丝上产生张力，而微管丝可以被感受到。 当微管遇到障碍物时，张力会增加，甚至更多。 电机被激活以绕过路障，或者，面临最高阻力的电机放开， 允许微管局部扩散并采取新的路径，电机之间的力共享至关重要。 这个想法是，多个电机可以让货物的速度在没有或存在的情况下大致保持恒定。 路障和弯路；然而，由于存在这些障碍，多个电机的力量叠加在一起，使得 货物顺利通过或绕过障碍物的例子包括： 可以打开和关闭的不同微管相关蛋白 (MAP)，以及肌动蛋白和中间体 细丝。 我们使用多种哺乳动物驱动蛋白或多种酵母动力蛋白，将来我们将使用多种 驱动蛋白和多种人类细胞质动力蛋白，该实验提出了这些后者如何或是否的问题 两个方向相反的电机系列相互竞争或合作，我们有初步数据。 多个驱动蛋白，发现它们是很好的共享者，并且动态地进出微管。 有多种酵母动力蛋白以及驱动蛋白和酵母动力蛋白的数据，我们发现分子马达。 “阻碍”和“驾驶”位置之间存在差异，这些位置会随着路障的变化而动态变化。 还模拟体内环境，例如，盐浓度和竞争细丝很高，或者 免费电机的可用性有限。 这里介绍的技术也是创新的。我们的技术将涉及测量单分子。 具有纳米分辨率的荧光；它将涉及基于以下参数的皮牛顿范围内的力测量： 使用具有蜘蛛丝张力的独特的蠕虫状 DNA 链或聚乙二醇来发出荧光 传感器；它将涉及测量多个单独运行但作用于单个货物的电机。