Chromosome biorientation requires Aurora B’s spatial separation from its outer kinetochore substrates but not its turnover at kinetochores

For correct chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (biorientation). For this, aberrant kinetochore–microtubule interaction must be resolved (error correction) by Aurora B kinase. Once biorientation is formed, tension is applied on kinetochore–microtubule interaction, stabilizing this interaction. The mechanism for this tension-dependent process has been debated. Here, we study how Aurora B localizations at different kinetochore sites affect the biorientation establishment and maintenance in budding yeast. Without the physiological Aurora B–INCENP recruitment mechanisms, engineered recruitment of Aurora B–INCENP to the inner kinetochore, but not to the outer kinetochore, prior to biorientation supports the subsequent biorientation establishment. Moreover, when the physiological Aurora B–INCENP recruitment mechanisms are present, an engineered Aurora B–INCENP recruitment to the outer kinetochore, but not to the inner kinetochore, during metaphase (after biorientation establishment) disrupts biorientation, which is dependent on the Aurora B kinase activity. These results suggest the spatial separation of Aurora B from its outer kinetochore substrates is required to stabilize kinetochore–microtubule interaction when biorientation is formed and tension is applied on this interaction. Meanwhile, Aurora B exhibits dynamic turnover on the centromere/kinetochore during early mitosis, a process thought to be crucial for error correction and biorientation. However, using the engineered Aurora B–INCENP recruitment to the inner kinetochore, we demonstrate that, even without such a turnover, Aurora B–INCENP can efficiently support biorientation. Our study provides important insights into how Aurora B promotes error correction for biorientation in a tension-dependent manner