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，长距离和速度， 几乎一样的移动时，一个简单的缓冲区，一直是神秘的。路障，绕道， 平均而言，稠密的环境显然不会减慢货物在细胞周围移动的速度。这里我们 报告了一个简单的机制，基于一种新型的体外力滑行试验，其中多个电机运行 同时在一个共同的货物和他们的力量联合收割机。货物是一种微管， 上面是一系列随机放置但相距很远的马达，这些马达通过弹簧固定在盖玻片上。货物的 位置和速度通过荧光测量;每个电机的力用皮牛顿测量 通过测量平衡位移，在数分钟内保持准确度。这是我们第一次 设法开发了一种定量测量多个电机的检测方法，而不是单个电机。 紧张是沟通的关键。一个马达在微管丝上产生张力， 同一个微管上的其他马达。当微管遇到障碍时，张力会增加， 发动机被激活以绕过路障。或者，面对最高阻力的电机放手， 允许微管局部扩散并采取新的路径。马达之间的力共享是至关重要的。 这个想法是，多个马达允许货物的速度大致恒定，无论是否存在。 路障和弯路;然而，有了这些障碍，多个马达的力量加在一起， 货物平稳地穿过或绕过障碍物。路障/绕道的例子包括 不同的微管相关蛋白（MAP），可以来和关闭，以及肌动蛋白和中间 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，后者的实验问的问题是，如何，或如果，这些 两个方向相反的电动机族相互竞争或合作。我们有初步的数据， 多个驱动蛋白，发现它们是很好的共享者，动态地进入和离开微管。我们也 有多种酵母动力蛋白的数据，以及驱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"驱动"位置之间变化，其作为路障的函数动态地改变。我们将 还模拟体内环境，其中，例如，盐浓度和竞争细丝高，或者 免费电动机的可用性是有限的。 这里介绍的技术也是创新的。我们的技术将包括测量单个分子 荧光与纳米分辨率;它将涉及测量的基础上，在皮牛顿范围内的力量 使用具有蜘蛛蚕张力的DNA或聚乙二醇的独特蠕虫状链的荧光 传感器;它将涉及测量多个电机，所有单独行动，但对一个单一的货物工作。

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.