Ensemble-level organization of human kinetochores and evidence for distinct tension and attachment sensors

Kinetochoresare multi-protein machines that form dynamic attachments to microtubules and generate the forces for chromosome segregation. High-fidelity is ensured because kinetochorescan monitor attachment status and tension, using this information to activate checkpoints and error correction mechanisms. To explore how kinetochoresachieve this we used two and three colour subpixel fluorescence localisation to define how six protein subunitsfrom the major kinetochorecomplexes CCAN, MIS12, NDC80, KNL1, RZZ and the checkpoint proteins Bub1and Mad2are organised in the human kinetochore. This reveals how the kinetochoreouter plate is a liquid crystal-like system with high nematic order and largely invariant to loss of attachment or tension except for two mechanical sensors. Firstly, Knl1 unravelling relays tension and secondly NDC80jack-knifes under microtubule detachment, with only the latter wired up to the checkpoint signalling system. This provides insight into how kinetochoresintegrate mechanical signals to promote error-free chromosome segregation.