An Arabidopsis Kinesin-14D motor is associated with midzone microtubules for spindle morphogenesis.

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.

Confocal Microscopic Assays of Mitotically Active Proteins in an Agrobacterial Infiltration-Based, Cell Division-Enabled Leaf System of Tobacco (Nicotiana benthamiana).

The production of tissues and organs in plants is brought about by mitotic cell divisions, starting from the zygote. Successful mitosis and cytokinesis harness the functional input of proteins that are expressed in cell cycle-dependent manners to regulate cytoskeletal reorganization and intracellular motility. Fluorescence microscopic assays of mitotically active proteins have been dependent on time-consuming transformation experiments in a host plant or cultured cells. To facilitate the detection and observation of cell cycle-dependent localization and dynamics of plant proteins, we demonstrate, in this chapter, a transiently induced cell division system in Nicotiana benthamiana, named the cell division-enabled leaf system (CDELS). Plasmid constructs which express the D-type cyclin along with a fluorescent fusion protein(s) of interest are delivered to the leaves of N. benthamiana by agrobacterial infiltration. Ectopic expression of cyclin D induces leaf epidermal cells to re-enter mitosis and subsequently cytokinesis, allowing the dynamic localization of fluorescent fusion protein(s) to be observed throughout the course of mitotic cell division using live-cell fluorescence microscopy. This effective approach not only allows one to detect mitotic activities of novel proteins but also record their dynamics and relationship with others during mitosis and cytokinesis in a greatly shortened period of time.