CCAN forms the spherical shell-like structure in interphase kinetochores

Summary The kinetochore is an essential structure for the faithful chromosome segregation during mitosis. The 16-protein complex known as the constitutive centromere-associated network (CCAN) localizes the kinetochore throughout the cell cycle and forms the basis of the kinetochore. Although a structural model of an individual CCAN unit has been proposed through cryo-electron microscopy analysis, little is known about how multiple CCAN units are organized within cells. Here, using in silico and in vivo analyses, we demonstrate that multiple CCAN units form a spherical, shell-like structure in interphase DT40 kinetochores. CENP-T and CENP-N localize to the CCAN shell, and the CENP-C cupin domain clusters at the center. Molecular dynamics simulations of CCAN assembly suggest that CENP-C contributes to the orientation of CCAN units, leading to the formation of a spherical shell-like structure via CENP-C oligomerization. Super-resolution imaging reveals the spherical shell-like structure at DT40 kinetochores, and knocking out CENP-C abolishes the structure, supporting the reliability of the predicted models. These findings provide important insights into the spatial organization of functional kinetochores within cells.