Single kinetochores execute an ordered series of molecular events as the spindle assembly checkpoint is silenced

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 reorganization 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 MPS1 retained. Coincidently, the NDC80 ensemble reconfigures, an event dependent on microtubule binding. Kinetochores next transition into a mature attachment state, marked by a switch-like recruitment of Astrin-SKAP. This then undergoes gradual further stabilization through NDC80 tail dephosphorylation. By preventing biorientation, we also define otherwise hidden kinetochore states involved in error correction cycles. These results provide a critical temporal framework for understanding the mechanisms of SAC silencing and error correction at single human kinetochores.