Microtubules guide Aurora B substrate geometries for accurate chromosome segregation.

Accurate chromosome segregation requires differential regulation of microtubule-binding substrates by Aurora B, the kinase subunit of the chromosomal passenger complex (CPC). How microtubules simultaneously up- and down-regulate Aurora B phosphorylation remains unclear. Devising a new cryo-electron microscopy workflow, we determined microtubule-bound structures of the CPC and key Aurora B substrates that resolve their phosphorylation sites, finding that microtubules can promote or restrict Aurora B-mediated phosphorylation depending upon binding geometry. The kinetochore Ndc80 complex oligomerizes on microtubules through multivalent interactions including its kinase recognition sites, sterically restricting kinase access to counteract phosphorylation-induced detachment. Attenuating this oligomerization compromised stable kinetochore-microtubule attachments and causes chromosome mis-segregation. Conversely, Aurora B recognition sites of the microtubule-depolymerase mitotic centromere-associated kinesin (MCAK) remain accessible on microtubules, explaining how microtubule-bound CPC can promote MCAK phosphorylation and inactivation. We propose that microtubule-guided substrate remodeling can serve as a general mechanism for controlling Aurora B-mediated phosphorylation during mitosis, which can coordinate diverse processes underlying faithful chromosome segregation.