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.