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

摘要

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.