Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

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Abstract
Centromere protein F (CENP‐F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore‐bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP‐F does not affect cell cycle progression in S and G2. In a subset of CENP‐F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis‐segregation. In contrast, the majority of CENP‐F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi‐oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP‐marked Mad1 reveals that throughout the mitotic delay some aligned, CENP‐F depleted kinetochores continuously recruit Mad1. Others rebind YFP‐Mad1 intermittently so as to produce ‘twinkling’, demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP‐F in efficient assembly of a stable microtubule–kinetochore interface.