Three-Dimensional Optical Tweezers Tracking Resolves Random Sideward Steps of the Kinesin-8 Kip3

The budding yeast kinesin-8 Kip3 is a highly processive motor protein that walks to the ends of cytoskeletal microtubules and shortens them in a collective manner. However, how exactly Kip3 reaches the microtubule end is unclear. Although rotations of microtubules in multimotored Kip3 gliding assays implied directed sideward switching between microtubule protofilaments, two-dimensional, single-molecule, optical-tweezers assays indicated that Kip3 randomly switched protofilaments. Here, we topographically suspended microtubules such that Kip3 motors could freely access the microtubules in three dimensions. Tracking single-motor-driven microspheres with a three-dimensional, zero-load, optical-tweezers-based force clamp showed that Kip3 switched protofilaments in discrete steps equally frequent in both directions. A statistical analysis confirmed the diffusive sideward motion of Kip3, consistent with the two-dimensional single-molecule results. Furthermore, we found that motors were in one of three states: either not switching protofilaments or switching between them with a slow or fast sideward-stepping rate. Interestingly, this sideward diffusion was limited to one turn, suggesting that motors could not cross the microtubule seam. The diffusive protofilament switching may enable Kip3 to efficiently bypass obstacles and reach the microtubule end for length regulation.