Distinctive Kinesin-14 Motors Associate with Midzone Microtubules to Construct Mitotic Spindles with Two Convergent Poles in Arabidopsis

Abstract Microtubule (MT) motors in the Kinesin-14 subfamily proliferated in photosynthetic organisms and they often incorporated sequences bearing novel structural features. To gain insights into the functions of diversified Kinesin-14 motors from an evolutionary perspective, we performed phylogenetic analyses across different eukaryotic kingdoms. Compared to fungi that have a single class of Kinesin-14, the early divergent protist Giardia possesses two classes and the motile green alga Chlamydomonas produces four classes (Kinesin-14A to Kinesin-14D). The fifth class Kinesin-14E first appeared among immotile green algae and the sixth Kinesin-14F emerged in mosses, concomitantly with the display of 3D growth. The conservation of Kinesin-14D from green algae prompted us to investigate its function in Arabidopsis in which three such motors functioned in cell cycle-dependent manners. They localized on selective spindle MTs and/or sometimes kinetochore-like structures, and later all became conspicuous on MT bundles in the spindle midzone following sister chromatid segregation. Genetic dissection of Kinesin-14D1 showed that its loss led to hypersensitivity to low doses of the MT-depolymerizing herbicide oryzalin. Kinesin-14D1 association with the midzone MTs in both prophase and mitotic spindles. The oryzalin treatment left behind discrete kinetochore fibers attached to randomly positioned chromosomes in the mitotic kinesin-14d1 cells but prevented the pole convergence of bipolar mitotic spindles. This function of Kinesin-14D1 in the spindle midzone is likely dependent on an MT-binding domain at the C-terminus to the catalytic motor domain. Therefore, our results revealed a novel Kinesin-14D-dependent mechanism that regulates the formation of bipolar spindle apparatus with converged acentrosomal poles.