Abstract
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.




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References
Andreassen PR, Margolis RL (1994) Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J Cell Biol 127:789–802
Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Duncan K, Wordeman L, Swedlow JR (2004) Aurora B regulates MCAK at the mitotic centromere. Dev Cell 6:253–268
Brito DA, Rieder CL (2006) Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr Biol 16:1194–1200
Cai S, O'Connell CB, Khodjakov A, Walczak CE (2009) Chromosome congression in the absence of kinetochore fibres. Nat Cell Biol 11:832–838
Cheeseman IM, Chappie JS, Wilson-Kubalek EM, Desai A (2006) The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127:983–997
Chen RH, Shevchenko A, Mann M, Murray AW (1998) Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J Cell Biol 143:283–295
DeLuca JG, Gall WE, Ciferri C, Cimini D, Musacchio A, Salmon ED (2006) Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 127:969–982
Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 161:267–280
Dujardin D, Wacker UI, Moreau A, Schroer TA, Rickard JE, De Mey JR (1998) Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment. J Cell Biol 141:849–862
Goshima G, Vale RD (2003) The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J Cell Biol 162:1003–1016
Johnson VL, Scott MI, Holt SV, Hussein D, Taylor SS (2004) Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression. J Cell Sci 117:1577–589
Kapoor TM, Lampson MA, Hergert P, Cameron L, Cimini D, Salmon ED, McEwen BF, Khodjakov A (2006) Chromosomes can congress to the metaphase plate before biorientation. Science 311:388–391
Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM (2004) Correcting improper chromosome-spindle attachments during cell division. Nat Cell Biol 6:232–237
Maffini S, Maia AR, Manning AL, Maliga Z, Pereira AL, Junqueira M, Shevchenko A, Hyman A, Yates JR, Galjart N, Compton DA, Maiato H (2009) Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux. Curr Biol 19(18):1566–1572
Maia AF, Lopes CS, Sunkel CE (2007) BubR1 and CENP-E have antagonistic effects upon the stability of microtubule–kinetochore attachments in Drosophila S2 cell mitosis. Cell Cycle 6:1367–1378
Mao Y, Abrieu A, Cleveland DW (2003) Activating and silencing the mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1. Cell 114:87–98
McEwen BF, Chan GK, Zubrowski B, Savoian MS, Sauer MT, Yen TJ (2001) CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells. Mol Biol Cell 12:2776–2789
McIntosh JR, Grishchuk EL, West RR (2002) Chromosome–microtubule interactions during mitosis. Annu Rev Cell Dev Biol 18:193–219
Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8:379–393
Pinsky BA, Kung C, Shokat KM, Biggins S (2006) The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores. Nat Cell Biol 8:78–83
Putkey FR, Cramer T, Morphew MK, Silk AD, Johnson RS, McIntosh JR, Cleveland DW (2002) Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E. Dev Cell 3:351–365
Schaar BT, Chan GK, Maddox P, Salmon ED, Yen TJ (1997) CENP-E function at kinetochores is essential for chromosome alignment. J Cell Biol 139:1373–1382
Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA, Medema RH (2000) Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat Cell Biol 2:672–676
Tanaka TU, Rachidi N, Janke C, Pereira G, Galova M, Schiebel E, Stark MJ, Nasmyth K (2002) Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell 108:317–329
Tanudji M, Shoemaker J, L'Italien L, Russell L, Chin G, Schebye XM (2004) Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay. Mol Biol Cell 15:3771–781
Vader G, Maia AF, Lens SM (2008) The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond. Cell Div 3:10
Weaver BA, Bonday ZQ, Putkey FR, Kops GJ, Silk AD, Cleveland DW (2003) Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss. J Cell Biol 162:551–563
Wood KW, Sakowicz R, Goldstein LS, Cleveland DW (1997) CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell 91:357–366
Yang Z, Kenny AE, Brito DA, Rieder CL (2009) Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments. J Cell Biol 186:675–684
Yao X, Abrieu A, Zheng Y, Sullivan KF, Cleveland DW (2000) CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint. Nat Cell Biol 2:484–491
Yasui Y, Urano T, Kawajiri A, Nagata K, Tatsuka M, Saya H, Furukawa K, Takahashi T, Izawa I, Inagaki M (2004) Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis. J Biol Chem 279:12997–13003
Yen TJ, Li G, Schaar BT, Szilak I, Cleveland DW (1992) CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature 359:536–539
Yucel JK, Marszalek JD, McIntosh JR, Goldstein LS, Cleveland DW, Philp AV (2000) CENP-meta, an essential kinetochore kinesin required for the maintenance of metaphase chromosome alignment in Drosophila. J Cell Biol 150:1–11
Acknowledgments
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).
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Communicated by A. Musacchio
Claudio E. Sunkel and Susanne M. A. Lens contributed equally.
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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)
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)
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)
Supplementary ESM 1
(JPEG 51 kb)
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)
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)
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)
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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Maia, A.F., Feijão, T., Vromans, M.J.M. et al. Aurora B kinase cooperates with CENP-E to promote timely anaphase onset. Chromosoma 119, 405–413 (2010). https://doi.org/10.1007/s00412-010-0265-x
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DOI: https://doi.org/10.1007/s00412-010-0265-x