Low kinetochore-microtubule occupancy leaves a mitotic memory by delaying checkpoint silencing.

The spindle assembly checkpoint (SAC) promotes faithful chromosome segregation by delaying mitosis until all kinetochores attach to spindle microtubules. However, unusually prolonged mitoses block daughter cell proliferation through a p53-dependent memory mechanism-the "mitotic stopwatch"-suggesting a selective pressure to avoid significant mitotic delays. Here, we show that microtubule occupancy at kinetochores is a cornerstone linking SAC silencing with mitotic duration and memory. By combining live-cell with super-resolution microscopy, photobleaching and laser microsurgery in Indian muntjac fibroblasts, we demonstrate that SAC silencing is gradual and confined to microtubule attachments within kinetochores. Augmin promotes timely SAC silencing with high microtubule occupancy at kinetochores, whereas MPS1/CDK1 inhibition silences the SAC irrespective of microtubule occupancy. Conversely, low microtubule occupancy delays SAC silencing, increases segregation errors and blocks daughter cell proliferation due to mitotic stopwatch surveillance. Thus, timely SAC silencing with high microtubule occupancy avoids "bad memories" of mitosis to allow daughter cell proliferation.