The unconventional TPX2 family protein TPXL3 regulates α Aurora kinase function in spindle morphogenesis in Arabidopsis

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.