Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint.
Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.
Holt
Sarah V
SV
Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
Vergnolle
Mailys A S
MA
Hussein
Deema
D
Wozniak
Marcin J
MJ
Allan
Victoria J
VJ
Taylor
Stephen S
SS
D002453:Cell Cycle; D002503:Centromere / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D023902:Chromosome Pairing / Q000502:physiology*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D008677:Metaphase; D008840:Microfilament Proteins; D008938:Mitosis; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D008941:Spindle Apparatus / Q000502:physiology*; D014162:Transfection; D014404:Tubulin / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C094772:centromere protein F; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases