, Volume 119, Issue 4, pp 405–413 | Cite as

Aurora B kinase cooperates with CENP-E to promote timely anaphase onset

  • André F. Maia
  • Tália Feijão
  • Martijn J. M. Vromans
  • Claudio E. Sunkel
  • Susanne M. A. LensEmail author


Error-free chromosome segregation requires that all chromosomes biorient on the mitotic spindle. The motor protein Centromere-associated protein E (CENP-E) facilitates chromosome congression by mediating the lateral sliding of sister chromatids along existing K-fibers, while the mitotic kinase Aurora B detaches kinetochore–microtubule interactions that are not bioriented. Whether these activities cooperate to promote efficient chromosome biorientation and timely anaphase onset is not known. We here show that the chromosomes that fail to congress after CENP-E depletion displayed high centromeric Aurora B kinase activity. This activity destabilized spindle pole proximal kinetochore–microtubule interactions resulting in a checkpoint-dependent mitotic delay that allowed CENP-E-independent chromosome congression, thus reducing chromosome segregation errors. This shows that Aurora B keeps the mitotic checkpoint active by destabilizing kinetochore fibers of polar chromosomes to permit chromosome congression in CENP-E-compromised cells and implies that this kinase normally prevents pole proximal syntelic attachments to allow CENP-E-mediated congression of mono-oriented chromosomes.


Electronic Supplementary Material Nocodazole Spindle Pole Mitotic Checkpoint Supplementary Movie 
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We thank A. Musacchio, N. Galjart, and D. Cleveland for sharing the reagents and H. Maiato, M. Tanenbaum, G. Kops, and R. Medema for comments on the manuscript. A.F.M. is supported by a Ph.D. fellowship from Fundação para a Ciência e a Tecnologia (FCT) of Portugal and by a short-term fellowship from EMBO. This work was supported by grants from FCT (PTDC/BIA-BCM/64224/2006, C.E.S.) and the Netherlands Organization for Scientific Research (Vidi 917.66.332, S.M.A.L).

Supplementary material

412_2010_265_Fig1_ESM.jpg (45 kb)
Supplementary Fig. 1

(a) Western blot for CENP-E. Different amounts of total protein was loaded of mock-transfected cells to determine the detection limit of the CENP-E Ab. CENP-E siRNA-transfected cells have at least five times less CENP-E protein than control cells. α-Tubulin was used as loading control. (b) Quantification of the number of CENP-E-positive cells observed by IF in control (siLuciferase) and CENP-E-depleted cells. Error bars represent standard deviation (SD). (c) Experimental setup used to analyze mitotic progression by time-lapse microscopy in HeLa cells stably expressing YFP-H2B. Cells were continuously filmed 7 h after the release of a thymidine block, where indicated DMSO was added to account for the solvent of compounds that were used. (d) Time spent in different mitotic phases in 20 randomly selected HeLa YFP-H2B cells. Bars denote time in prometaphase (blue), time in metaphase (red), and time of anaphase onset (yellow) (JPEG 45 kb)

412_2010_265_Fig2_ESM.jpg (41 kb)
Supplementary Fig. 2

(a, b) Immunofluorescence images showing clear kinetochore localization of Mad1 (a) and CLIP-170 (b) in the unaligned chromosomes (insets, higher magnification) with almost complete absence on the aligned chromosomes. Scale bars are 5 μm (JPEG 41 kb)

412_2010_265_Fig3_ESM.jpg (26 kb)
Supplementary Fig. 3

(a) Immunofluorescence images of control siRNA-treated cells showing Mad1 localization (green) with or without 35-min treatment with ZM447439. Cells were also treated with the microtubule depolymerizing drug nocodazole. DNA is shown in blue and CREST in red. Scale bar is 5 μm. (b) Bar graph plotting the fluorescence ratio of Mad1 to CREST signal under control (DMSO) or ZM447439 treatment. Error bars represent SD (JPEG 25 kb)

412_2010_265_Fig4_ESM.jpg (52 kb)
Supplementary ESM 1

(JPEG 51 kb)

Supplementary movies 1 and 2

Animated time-lapse images of a control (siLuciferase) HeLa cell stably expressing YFP-H2B. Images were taken every 3 min. Overlay of DIC image (gray) and YFP-H2B (green), movie 1. YFP-H2B (gray), movie 2 (MPG 2112 kb)

(1.89 MB)

Supplementary movies 3 and 4

Animated time-lapse images of a CENP-E-depleted HeLa cell stably expressing YFP-H2B. Phenotype a. Note that the cell only goes into anaphase after the last unaligned chromosome reaches the metaphase plate. Images were taken every 3 min. Overlay of DIC image (gray) and YFP-H2B (green), movie 3. YFP-H2B (gray), movie 4 (MPG 1940 kb)

(5.43 MB)

Supplementary movies 5 and 6

Animated time-lapse images of a CENP-E-depleted HeLa cell stably expressing YFP-H2B. Phenotype b. Note that the cell goes into anaphase with an unaligned chromosome. Images were taken every 3 min. Overlay of DIC image (gray) and YFP-H2B (green), movie 5. YFP-H2B (gray), movie 6 (MPG 5566 kb)

(2.96 MB)

Supplementary movies 7 and 8

Animated time-lapse images of a CENP-E-depleted HeLa cell stably expressing YFP-H2B. A black frame indicates that ZM447439 was added to the medium. Note that anaphase starts with several unaligned chromosomes. Images were taken every 3 min. Overlay of DIC image (gray) and YFP-H2B (green), movie 7. YFP-H2B (gray), movie 8 (MPG 3034 kb)

(3.22 MB)


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • André F. Maia
    • 1
  • Tália Feijão
    • 1
  • Martijn J. M. Vromans
    • 2
  • Claudio E. Sunkel
    • 1
    • 3
  • Susanne M. A. Lens
    • 2
    Email author
  1. 1.IBMC—Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  2. 2.Department of Medical OncologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  3. 3.ICBAS—Instituto de Ciências Biomédicas Abel Salazar da Universidade do PortoPortoPortugal

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