Analysis scripts for the manuscript "Spindle checkpoint silencing at kinetochores with submaximal microtubule occupancy" by Banafsheh Etemad, Abel Vertesy, Timo E.F. Kuijt, Carlos Sacristan, Alexander van Oudenaarden, Geert J.P.L. Kops.
The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. SAC proteins are shed from kinetochores once stable attachments are achieved. Human kinetochores consist of hundreds of SAC protein recruitment modules and bind up to 20 microtubules, raising the question how the SAC responds to intermediate attachment states. We show that the ‘RZZS-MAD1/2’ module of the SAC is removed from kinetochores at low microtubule occupancy and remains absent at higher occupancies, while the ‘BUB1/R1’ module is retained at substantial levels irrespective of attachment states. These behaviours reflect different silencing mechanisms: while BUB1 displacement is almost fully dependent on MPS1 inactivation, MAD1 displacement is not. Artificially tuning the affinity of kinetochores for microtubules further shows that ~50% occupancy is sufficient to shed MAD2 and silence the SAC. Kinetochores thus responds as a single unit to shut down SAC signaling at submaximal occupancy states, but retains one SAC module. This may ensure continued SAC silencing on kinetochores with fluctuating occupancy states while maintaining the ability for fast SAC re-activation.