The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls mitosis through assembly of a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1, 2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4-9]. Through gene editing and live-cell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10-13] once Mps1 phosphorylates the N-terminus of Rod. Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells. We conclude that RZZ drives structural changes in the outer kinetochore that facilitate chromosome bi-orientation and chronic SAC transduction, a key determinant of cytotoxicity during anti-mitotic drug therapy [14-16].