Arabidopsis TPXL Proteins Mediate a Selective Aurora Kinase-Microtubule Association During Cell Division.

Microtubule organization during cell division requires coordinated regulation by microtubule-associated proteins and protein kinase signaling pathways. In animals, TPX2 (Targeting Protein for Xklp2) regulates spindle assembly by binding to and activating Aurora A kinase. In Arabidopsis, individual TPX2-like (TPXL) proteins have been implicated in Aurora-associated functions, but whether the TPXL family members share common microtubule-associated properties and how TPXL-Aurora interactions differ across the family remain incompletely understood. Here, we investigated the structural and cellular relationships among the eight Arabidopsis TPX2-like proteins (TPXL1-TPXL8), microtubules, and Aurora kinases. AlphaFold3-based structural prediction indicated that specific interaction sites between TPXL proteins and tubulins were revealed at the three-dimensional structure and atomic level, suggesting that all TPXL proteins contain a conserved TPX2 domain contributing to their predicted tubulin association. Transient expression analyses showed that TPXL1-TPXL8 localized to microtubule arrays during both interphase and cell division. Although microtubule-associated domain is predicted to be conserved across the family, individual TPXL members exhibit distinct localization patterns on spindles and phragmoplasts, suggesting potential functional specialization. Further structural prediction and experimental validation revealed that only TPXL2, TPXL3, TPXL4, and TPXL8 interacted with AUR1 (Aurora 1) and AUR2, whereas no detectable interactions were observed for the other TPXL members or for any TPXL protein with AUR3. Structural prediction of TPXL-AUR1-tubulin complexes were consistent with a possible arrangement in which TPXL proteins contact both AUR1 and tubulin. Together, our findings suggest that Arabidopsis TPXL proteins have broad microtubule association and are coupled with selective Aurora kinase interaction to coordinate microtubule organization, providing a framework for future functional analysis of TPXL-Aurora-microtubule association during plant cell division.