Conway, C. C.; Germanova, T.; Toral-Perez, S.; Coates, C.; Pines, J.; Burroughs, N. J.; McAinsh, A. D.

The Spindle Assembly Checkpoint (SAC) delays anaphase onset until all kinetochores are stably attached to microtubules, thus promoting error-free chromosome segregation. Multiple molecular events are implicated in SAC silencing, including removal of phospho-marks, protein (un)binding, and structural reorganisation of the kinetochore - but we currently lack a quantitative map of how these events unfold through time. Here, we use the levels of the checkpoint protein MAD2 to create a pseudo-timeline of SAC silencing at single kinetochores. We demonstrate how silencing proceeds through an ordered series of molecular events where MAD2-Spindly unbinds first and then the KNL1 catalytic platform disassembles, with a pool of active MPS1 retained. Coincidently, the NDC80 ensemble reconfigures in response to high microtubule occupancy. Kinetochores next switch into a mature attachment state that then undergoes gradual further stabilisation through NDC80 tail dephosphorylation. By preventing biorientation, we also define otherwise hidden kinetochore states involved in error correction cycles. This includes a \"poised\" state which we propose allows for error correction and rapid reactivation of the SAC. These results provide a critical temporal framework for understanding the mechanisms of SAC silencing and error correction at single human kinetochores.