Three-Dimensional Motility of the Highly Processive Kinesin-8 Along the Microtubule Lattice

Abstract

During mitosis, kinesin-8 motors play a key role in regulating the spindle length based to their depolymerization activity at microtubule plus-ends. In order to reach the plus-ends of microtubules, kinesin-8 motors show superprocessive motility. However, the mechanism confering such high motor processivity even on crowded microtubules in the cytoplasm is not well understood. To gain insight into the stepping mechanism of kinesin-8, we explored the three-dimensional motility of yeast kinesin-8, Kip3, along the microtubule lattice in vitro. First, we performed microtubule gliding motility assays on surface coated with Kip3 motors and measured the rotations of gliding microtubules around their longitudinal axis using rhodamine speckled microtubules in combination with fluorescence-interference contrast microscopy [1]. We observed rotations with periodicities significantly smaller than the microtubule supertwist, indicating that the motors do not follow individual protofilaments but rather switch protofilaments stochastically in one direction [2]. Finally, we confirmed this hypothesis by 3D single-molecule stepping assays along freely suspended microtubules by 2D tracking of QDot-conjugated Kip3 motors in combination with parallax [3], a dual-focus imaging technique that provides nanometer information in z-direction.1. Mitra A, Ruhnow F, Nitzsche B, Diez S. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces. PLoS One. 2015.2. Bormuth V, Nitzsche B, Ruhnow F, Mitra A, Storch M, Rammner B, Howard J, Diez S. The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left. Biophys J. 2012.3. Sun Y, McKenna JD, Murray JM, Ostap EM, Goldman YE. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 2009.