The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left

Abstract

Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.

Figure 1

Monitoring Kip3-driven microtubule rotations in gliding motility assays. (A) Schematic of the experimental setup. Imaging is performed on top of a reflective silicon surface using fluorescence interference contrast (FLIC) microscopy (2). (B) Maximum projection of the fluorescence signal of a microtubule-attached quantum dot in the Kip3 gliding motility assay. (C) FLIC intensity (red) and lateral distance from the microtubule path (blue) of the quantum dot shown in panel B versus traveled distance along the microtubule path. The periodic FLIC signal is indicative of repeated up- and down-motion. (D) Schematic of the deduced Kip3 path (red) in comparison to the protofilament axis (green) on a 14-protofilament microtubule.

Figure 2

Virtual three-dimensional reconstruction of Kip3 stepping. (A) Tubulin dimer: composed of alpha-tubulin (α) and beta-tubulin (β) monomers, with the unstructured surface-exposed E-Hooks (e). Kip3 front head: Shown with undocked neck linker (U) and following coiled-coil (cc) dimerization domain. Kip3 rear head: Shown with docked (D) and undocked (U) neck linker parts. (B) Illustration of different Kip3 configurations bound with both heads to adjacent tubulin dimers (first heptad repeat of the coiled coil region is artificially unfolded to illustrate all binding configurations). (C) Estimated three-dimensional distances between the positions where the neck linkers protrude from the motor heads, respecting the volumes of the heads (nomenclature as in panel B). For comparison, the direct distances between the tubulin dimers are given.