Cells coordinate diverse events at anaphase onset, including separase activation, cohesin cleavage, chromosome separation, and spindle reorganization. Regulation of the XMAP215 family member and microtubule polymerase, Stu2, at the metaphase-anaphase transition determines a redistribution from kinetochores to spindle microtubules. We show that cells modulate Stu2 kinetochore-microtubule localization by Polo-like kinase1/Cdc5-mediated phosphorylation of T866, near the Stu2 C-terminus, thereby promoting dissociation from the kinetochore Ndc80 complex. Cdk/Cdc28 likely primes Cdc5:Stu2 interaction. Cdc28 activity is also required for Stu2 nuclear import. PP2ACdc55 actively opposes Cdc5 activity on Stu2T866 during metaphase. This counter-regulation allows for switch-like redistribution of Stu2pT866 at anaphase onset when separase inhibits PP2ACdc55. Blocking Stu2T866 phosphorylation disrupts anaphase spindle progression, and we infer that PP2ACdc55 regulates the mitotic spindle by dephosphorylating multiple MAPs, including Stu2. These data support a model in which increased phosphorylation at anaphase onset results from phosphatase inhibition and point to a larger regulatory network that facilitates rapid cytoskeletal modulation required for anaphase spindle function.

TOG family proteins, including the budding yeast Stu2, are essential for the formation of a functional mitotic spindle. Across all eukaryotes, the described functions of this family depend on two microtubule binding elements: TOG domain arrays, and a basic linker domain important for binding the microtubule lattice. Consistently, we find here that Stu2’s basic linker is required for its ability to regulate microtubules in vitro, including stimulating microtubule growth, shrinkage, and catastrophe. We furthermore define a region contained within Stu2’s basic linker domain as its nuclear localization sequence, and identify phospho-regulation that promotes mitosis-specific nuclear import. Surprisingly, directing nuclear localization is the only function contained within Stu2’s basic linker that is required for cell viability, indicating that microtubule lattice binding is not required for Stu2’s essential function. Considering that lattice binding is required to stimulate microtubule polymerization and depolymerization in vitro, these established activities are unlikely to be the essential functions carried out by Stu2 in the cell’s nucleus.SUMMARY Stu2 is a TOG family protein that performs numerous microtubule regulatory functions in the cell. Here we show that Stu2’s nuclear localization is essential for cell viability. Surprisingly, its required nuclear function is distinct from its canonical activities regulating microtubules.### Competing Interest StatementThe authors have declared no competing interest.

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these ‘bioriented’ sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.