KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores

The spindle assembly checkpoint (SAC) ensures the accurate segregation of sister chromatids during mitosis. Activation of the SAC occurs through a series of ordered molecular events that result in recruitment of Mad1:Mad2 complexes to improperly attached kinetochores. The current model involves sequential phospho-dependent recruitment of Bub3:Bub1 to KNL1 followed by binding of Mad1:Mad2 to Bub1. Here, we show in non-transformed diploid human cells that the KNL1-Bub3-Bub1 (KBB) pathway is required during normal mitotic progression when kinetochores are misaligned but is nonessential for SAC activation and Mad2 loading when kinetochores are unattached from microtubules. We provide evidence that the Rod-ZW10-Zwilch (RZZ) complex is necessary to recruit Mad1:Mad2 to, and delay anaphase onset in response to, unattached kinetochores independently of the KBB pathway. These data suggest that the KBB and RZZ complexes provide two distinct kinetochore receptors for Mad1:Mad2 and reveal mechanistic differences between SAC activation by unattached and improperly attached kinetochores. [Display omitted] •KNL1 is required for SAC activation in human cells with misaligned kinetochores•RZZ is required to recruit Mad1:Mad2 complexes to unattached kinetochores•RZZ activates the SAC in cells with unattached kinetochores, independently of KNL1•RZZ and KBB are two distinct kinetochore receptors for Mad1:Mad2 at kinetochores Recruitment of Mad1:Mad2 complexes to unattached or misaligned kinetochores is a key step in generating the checkpoint signal that inhibits anaphase onset. Silió et al. show that human kinetochores utilize at least two different pathways to recruit Mad1:Mad2 complexes and that these pathways respond to distinct kinetochore-microtubule attachment problems.