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ABSTRACT Actin microfilaments (F-actin) serve as the track for directional movement of organelles in plant cells. In actively growing plant cells, F-actin often form robust bundles that trespass the cellular dimension. To test how the F-actin network was employed for peroxisome movement, we wished to disturb actin organization by genetically compromising the function of villin (VLN) proteins that serve as the primary bundling factor inArabidopsis thalianacells. To do so, we isolated T-DNA insertional mutants in threeVLNgenes that were most actively expressed in vegetative tissues. We found that thevln4mutation greatly enhanced the growth defects caused by thevln2 vln3double mutant as thevln2 vln3 vln4triple mutant had a great reduction of organ growth and formed heavily deformed tissues. Both VLN2 and VLN4 proteins were detected on bundled F-actin filaments. Compared to the wild-type cells, the double and triple mutants exhibited progressively reduction of stable F-actin bundles and had fine F-actin filaments undergo rapid remodeling. The defective F-actin network did not prevent peroxisomes from taking on both rapid and slow movements along the F-actin tracks. However, we found that compromised F-actin bundling caused significant reductions in the speed of peroxisome movement and the displacement distance of peroxisome positions. Using a correlation analysis method, we also demonstrated that the complex heterogeneous peroxisome movement may be classified into clusters reflecting the directionality of peroxisome movement. The triple mutant suffered from a significant reduction of peroxisomes exhibiting long-range and linear movement. Our results provided insights into how VLN-dependent F-actin organization was coupled with the complex patterns of peroxisome movement. 

The acentrosomal spindle apparatus has kinetochore fibers organized and converged toward opposite poles; however, mechanisms underlying the organization of these microtubule fibers into an orchestrated bipolar array were largely unknown. Kinesin-14D is one of the four classes of Kinesin-14 motors that are conserved from green algae to flowering plants. In Arabidopsis thaliana, three Kinesin-14D members displayed distinct cell cycle-dependent localization patterns on spindle microtubules in mitosis. Notably, Kinesin-14D1 was enriched on the midzone microtubules of prophase and mitotic spindles and later persisted in the spindle and phragmoplast midzones. The kinesin-14d1 mutant had kinetochore fibers disengaged from each other during mitosis and exhibited hypersensitivity to the microtubule-depolymerizing herbicide oryzalin. Oryzalin-treated kinesin-14d1 mutant cells had kinetochore fibers tangled together in collapsed spindle microtubule arrays. Kinesin-14D1, unlike other Kinesin-14 motors, showed slow microtubule plus end-directed motility, and its localization and function were dependent on its motor activity and the novel malectin-like domain. Our findings revealed a Kinesin-14D1-dependent mechanism that employs interpolar microtubules to regulate the organization of kinetochore fibers for acentrosomal spindle morphogenesis.