Reconstitution of Collective Transport by Defined Numbers of Kinesin-1 and Kinesin-14, Two Opposing Motor Proteins

Abstract

Intracellular transport is usually driven by the collective operation of molecular motors that move along cytoskeletal filaments. Recent studies have reported that the movements include dynamic changes in velocity and direction along the filaments, which may be explained as a result of interplay between teams of opposing motors. In these movements, the number of participating motors is so small that its stochastic behavior dominates. Therefore, to reveal the mechanisms of motor coordination, it is essential to build and test mechanical models that explicitly incorporate the number and geometry of engaged motors. Here, we develop an experimental system to control the number and geometry of motors, i.e. 1, 2, 3, and 4 molecules, at specific sites on a single DNA scaffold. The covalent bonds throughout the whole complex allow us to precisely quantify the number of engaged molecules by SDS-PAGE, and ensure the stable linkages between motors for motion tracking and force measurement. First, we constructed complexes that engage teams of either kinesin-1 or kinesin-14. The travel distances of both kinesins were greatly extended when the number of molecules was increased, whereas the velocities were only slightly affected. Next, we linked together two opposing kinesin motors, kinesin-1 and kinesin-14, by a single molecular scaffold, which leads to a tug-of-war on microtubules. In the tug-of-war, reversals of direction and periodical, step-like movements were frequently observed. The combination of quantitative in vitro and in silico experiments using this simplified system will provide fundamental knowledge of motor coordination.