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Abstract Spindle assembly in vertebrates requires the Aurora kinase, which is targeted to microtubules and activated by TPX2 (Targeting Protein of XKLP2). In Arabidopsis (Arabidopsis thaliana), TPX2-LIKE 3 (TPXL3), but not the highly conserved TPX2, is essential. To test the hypothesis that TPXL3 regulates the function of α Aurora kinase in spindle assembly, we generated transgenic Arabidopsis lines expressing an artificial microRNA targeting TPXL3 mRNA (amiR-TPXL3). The resulting mutants exhibited growth retardation, which was linked to compromised TPXL3 expression. In the mutant cells, α Aurora was delocalized from spindle microtubules to the cytoplasm, and spindles were assembled without recognizable poles. A functional TPXL3-GFP fusion protein first prominently appeared on the prophase nuclear envelope. Then, TPXL3-GFP localized to spindle microtubules (primarily toward the spindle poles, like γ-tubulin), and finally to the re-forming nuclear envelope during telophase and cytokinesis. However, TPXL3 was absent from phragmoplast microtubules. In addition, we found that the TPXL3 N-terminal Aurora-binding motif, microtubule-binding domain, and importin-binding motif, but not the C-terminal segment, were required for its mitotic function. Expression of truncated TPXL3 variants enhanced the defects in spindle assembly and seedling growth of amiR-TPXL3 plants. Taken together, our findings uncovered the essential function of TPXL3, but not TPX2, in targeting and activating α Aurora kinase for spindle apparatus assembly in Arabidopsis. 

Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana , the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo . The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.