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

摘要 意义：细胞质货物如何在拥挤的细胞内移动，在很长的距离和速度下 几乎和在一个简单的缓冲区中移动时一样，长期以来一直是个谜。路障、弯路和 平均而言，密集的环境显然不会减缓货物在细胞内移动的速度。在这里我们 报告一种简单的机制，基于一种新型的体外力量滑动测试，其中多个马达运行 同时在一个共同的货物和他们的力量结合。货物是运输的微管。 上面是一系列随机放置但相距很远的电机，这些电机固定在盖子上，穿过弹簧。货物是 位置和速度是通过荧光测量的；每个马达的力是用皮牛顿测量的 通过测量偏离平衡的位移，在多分钟内保持精度。第一次，我们有了 成功地开发了一种定量测量多台发动机的方法，而不是单一的发动机。 紧张是沟通的关键。一个马达在微管细丝上产生张力，由 同一微管上的其他马达。当微管面对障碍物时，张力就会增加。 马达被激活以绕过路障。或者，面对最高阻力的电机松开， 允许微管局部扩散并走一条新的路径。电机之间的力的分配至关重要。 其想法是，多个马达允许货物的速度在没有或存在的情况下大致恒定 路障和弯路；然而，有了这些障碍，多个马达的力加在一起，就可以 货物顺利通过或绕过障碍物。路障/绕道的例子包括 不同的微管相关蛋白(MAP)可以开启和关闭，以及肌动蛋白和中间体 细丝。 我们使用多个哺乳动物动蛋白或多个酵母动力蛋白，未来我们将使用多个 激动素和多个人类胞浆动力蛋白，后者的实验询问了这些是如何或是否 两个方向相反的马达家族相互竞争或合作。我们有初步数据显示 多种动蛋白，并发现它们是很好的共享者，并动态地在微管上来来去去。我们也 有多种酵母动力蛋白的数据，以及动蛋白和酵母动力蛋白的数据。我们发现分子马达 在“阻碍”和“驾驶”位置之间变化，随着路障的变化而动态变化。我们会 也可以模拟体内环境，例如，盐浓度和竞争的细丝很高，或者 免费马达的供应是有限的。 这里介绍的技术也是创新的。我们的技术将包括测量单分子 纳米分辨率的荧光；它将涉及测量皮牛顿范围内的力，基于 利用独特的蠕虫状DNA链或聚乙二醇链与蜘蛛丝张力进行荧光 传感器；它将涉及测量多个马达，这些马达都单独作用于单个货物。

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.