Kinetochore Homeostasis is Maintained by Coordinated Chromatin Stabilization and Soluble Buffering

SUMMARY Faithful chromosome segregation requires the coordinated assembly and maintenance of kinetochore complexes. However, the homeostatic mechanisms that maintain these multi-subunit assemblies remain unclear. CENP-T is a chromatin-bound linker that recruits the outer kinetochore modules via the CENP-T-W-S-X complex to form a functional kinetochore. Although CENP-T must be continuously replenished, how its steady-state levels are maintained is unknown. Here we demonstrate that CENP-T homeostasis is actively sustained by two spatially distinct pathways. Using covalent pulse-labeling, we find that CENP-T undergoes cell-cycle-coupled turnover rather than being stably inherited. In situ proximity labeling of chromatin-bound and soluble pools identifies regulators of CENP-T stability, including an unexpected role for the CENP-O complex in stabilizing soluble CENP-T. Mechanistically, CENP-S-X stabilizes chromatin-bound CENP-T through DNA-binding, whereas the CENP-O complex buffers a replenishment-competent CENP-T pool. Together, these conserved, spatially compartmentalized pathways establish an actively maintained homeostatic system that ensures robust kinetochore assembly and faithful chromosome segregation.