Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F.
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.
Bomont
Pascale
P
Department of Cellular and Molecular Medicine and Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA 92093, USA.
Maddox
Paul
P
Shah
Jagesh V
JV
Desai
Arshad B
AB
Cleveland
Don W
DW
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008840:Microfilament Proteins; D008870:Microtubules / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C094772:centromere protein F