Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint

• Published in: Journal of cell science Vol. 118; no. 20; pp. 4889 - 4900
• Main Authors: Holt, Sarah V, Vergnolle, Mailys A. S, Hussein, Deema, Wozniak, Marcin J, Allan, Victoria J, Taylor, Stephen S
• Format: Journal Article
• Language: English
• Published: England 15.10.2005
• Subjects: Cell Cycle; Centromere - physiology; Chromosomal Proteins, Non-Histone - deficiency; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Chromosome Pairing - physiology; Gene Silencing; HeLa Cells; Humans; Kinetochores - metabolism; Metaphase; Microfilament Proteins; Mitosis; Protein Kinases - metabolism; Protein-Serine-Threonine Kinases; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Spindle Apparatus - physiology; Transfection; Tubulin - metabolism
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.02614

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.