, Volume 118, Issue 1, pp 71–84 | Cite as

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres

  • Leena J. Ahonen
  • Anu M. Kukkonen
  • Jeroen Pouwels
  • Margaret A. Bolton
  • Christopher D. Jingle
  • P. Todd Stukenberg
  • Marko J. Kallio
Research Article


Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.



This work was supported by grants to MJK (Marie Curie EXT 002697, Academy of Finland 8206930), to PTS (R01GM063045-06), and Turku Graduate School of Biomedical Sciences. We thank Erich Nigg and Ulf Klein for sending GFP-hIncenp plasmid, David Wotton for providing pCS2 + YFP, and AstraZeneca Pharmaceuticals for providing ZM447439. We thank Jouko Sandholm at the Turku Centre for Biotechnology for his kind help with the photobleaching experiments.

Supplementary material

412_2008_178_MOESM1_ESM.doc (12 kb)
Table S1 Turnover of GFP-hIncenp in LLC-PK cells after various drug treatments (DOC 11.5 KB)
412_2008_178_MOESM2_ESM.jpg (1.7 mb)
Fig. S1 a Immunofluorescence images of fixed Xeno S3 cells stained with Incenp-ab. The antibody stains inner centromeres from prophase to metaphase, spindle microtubules in anaphase, and the midbody in telophase. When injected into metaphase (b) or anaphase cells (c), the Incenp-ab binds to inner centromeres or midzone microtubules, respectively. Cells were injected, fixed, and stained for tubulin (red in the overlay, one selected focal plane is shown), Incenp-ab (green), and DNA (blue, DAPI). Insets show magnified views of the metaphase plate and the spindle midzone. (de) Incenp-ab-injected Xeno S3 cells undergo a forced mitotic exit despite nocodazole or taxol-induced mitotic arrest. f Incenp-ab-injected Xeno S3 cells remain arrested at M phase in the presence of proteasome inhibitor MG312. Scale bars = 10 µm (JPEG 1.73 MB) (8.1 mb)
Video 1 Xeno S3 cell injected with Incenp-ab at prophase (MOV 8.07 MB). (4.6 mb)
Video 2 Xeno S3 cell injected with Incenp-ab at prophase (MOV 4.61 MB). (619 kb)
Video 3 GFP-tubulin expressing Xeno S3 cell injected with Incenp-ab at metaphase (MOV 619 KB). (1.4 mb)
Video 4 Recovery of photobleached GFP-xIncenp at the centromeres of a Xeno S3 cell in the presence of MG132 (MOV 1.36 MB). (1.3 mb)
Video 5 Recovery of photobleached GFP-xIncenp at the centromeres of a Xeno S3 cell after Incenp-ab injection in the presence of MG132 (MOV 1.30 MB). (2.7 mb)
Video 6 Recovery of photobleached xAurora B-YFP at the centromeres of a Xeno S3 cell in the presence of MG132 (MOV 2.70 MB). (2.1 mb)
Video 7 Recovery of photobleached xAurora B-YFP at the centromeres of a Xeno S3 cell after Incenp-ab injection in the presence of MG132 (MOV 2.08 MB). (1.6 mb)
Video 8 Xeno S3 cell injected with Incenp-ab at the onset of anaphase (MOV 1.56 MB). (1.3 mb)
Video 9 Xeno S3 cell injected with Incenp-ab 1 min after the onset of anaphase (MOV 1.28 MB). (3.2 mb)
Video 10 Xeno S3 cell injected with Incenp-ab 3 min after the onset of anaphase (MOV 3.23 MB). (3.7 mb)
Video 11 Xeno S3 cell injected with Incenp-ab 5 min after the onset of anaphase (MOV 3.67 MB). (507 kb)
Video 12 GFP-tubulin expressing Xeno S3 cell injected with Incenp-ab at early anaphase (MOV 506 KB). (662 kb)
Video 13 GFP-tubulin expressing Xeno S3 cell injected with control buffer at early anaphase (MOV 662 KB).


  1. Adams RR, Wheatley SP, Gouldsworthy AM, Kandels-Lewis SE, Carmena M, Smythe C, Gerloff DL, Earnshaw WC (2000) INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr Biol 10:1075–1078PubMedCrossRefGoogle Scholar
  2. Adams RR, Maiato H, Earnshaw WC, Carmena M (2001) Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J Cell Biol 153:865–880PubMedCrossRefGoogle Scholar
  3. Beardmore VA, Ahonen LJ, Gorbsky GJ, Kallio MJ (2004) Survivin dynamics increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity. J Cell Sci 117:4033–4042PubMedCrossRefGoogle Scholar
  4. Bishop JD, Schumacher JM (2002) Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B Kinase stimulates Aurora B kinase activity. J Biol Chem 277:27577–27580PubMedCrossRefGoogle Scholar
  5. Bolton MA, Lan W, Powers SE, McCleland ML, Kuang J, Stukenberg PT (2002) Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation. Mol Biol Cell 13:3064–3077PubMedCrossRefGoogle Scholar
  6. Brust-Mascher I, Civelekoglu-Scholey G, Kwon M, Mogilner A, Scholey JM (2004) Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation. Proc Natl Acad Sci U S A 101:15938–15943PubMedCrossRefGoogle Scholar
  7. Carvalho A, Carmena M, Sambade C, Earnshaw WC, Wheatley SP (2003) Survivin is required for stable checkpoint activation in taxol-treated HeLa cells. J Cell Sci 116:2987–2998PubMedCrossRefGoogle Scholar
  8. Cimini D, Wan X, Hirel CB, Salmon ED (2006) Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr Biol 16:1711–1718PubMedCrossRefGoogle Scholar
  9. Cooke CA, Heck MM, Earnshaw WC (1987) The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol 105:2053–2067PubMedCrossRefGoogle Scholar
  10. Delacour-Larose M, Molla A, Skoufias DA, Margolis RL, Dimitrov S (2004) Distinct dynamics of Aurora B and Survivin during mitosis. Cell Cycle 3:1418–1426PubMedGoogle Scholar
  11. Delacour-Larose M, Thi MN, Dimitrov S, Molla A (2007) Role of survivin phosphorylation by aurora B in mitosis. Cell Cycle 6:1878–1885PubMedGoogle Scholar
  12. DeLuca JG, Gall WE, Ciferri C, Cimini D, Musacchio A, Salmon ED (2006) Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 127:969–982PubMedCrossRefGoogle Scholar
  13. 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 161:267–280PubMedCrossRefGoogle Scholar
  14. Gadea BB, Ruderman JV (2005) Aurora kinase inhibitor ZM447439 blocks chromosome-induced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts. Mol Biol Cell 16:1305–1318PubMedCrossRefGoogle Scholar
  15. Giet R, Glover DM (2001) Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J Cell Biol 152:669–682PubMedCrossRefGoogle Scholar
  16. Girdler F, Gascoigne KE, Eyers PA, Hartmuth S, Crafter C, Foote KM, Keen NJ, Taylor SS (2006) Validating Aurora B as an anti-cancer drug target. J Cell Sci 119:3664–3675PubMedCrossRefGoogle Scholar
  17. Gorbsky GJ (2001) The mitotic spindle checkpoint. Curr Biol 11:R1001–1004PubMedCrossRefGoogle Scholar
  18. Guse A, Mishima M, Glotzer M (2005) Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis. Curr Biol 15:778–786PubMedCrossRefGoogle Scholar
  19. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T, Graham JA, Demur C, Hercend T, Diu-Hercend A, Su M, Golec JM, Miller KM (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 10:262–267PubMedCrossRefGoogle Scholar
  20. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM (2003) The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 161:281–294PubMedCrossRefGoogle Scholar
  21. Honda R, Korner R, Nigg EA (2003) Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis. Mol Biol Cell 14:3325–3341PubMedCrossRefGoogle Scholar
  22. Howell BJ, Moree B, Farrar EM, Stewart S, Fang G, Salmon ED (2004) Spindle checkpoint protein dynamics at kinetochores in living cells. Curr Biol 14:953–964PubMedCrossRefGoogle Scholar
  23. Jeyaprakash AA, Klein UR, Lindner D, Ebert J, Nigg EA, Conti E (2007) Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together. Cell 131:271–285PubMedCrossRefGoogle Scholar
  24. Kagey MH, Melhuish TA, Wotton D (2003) The polycomb protein Pc2 is a SUMO E3. Cell 113:127–137PubMedCrossRefGoogle Scholar
  25. Kaitna S, Mendoza M, Jantsch-Plunger V, Glotzer M (2000) Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis. Curr Biol 10:1172–1181PubMedCrossRefGoogle Scholar
  26. Kallio MJ, Beardmore VA, Weinstein J, Gorbsky GJ (2002a) Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells. J Cell Biol 158:841–847PubMedCrossRefGoogle Scholar
  27. Kallio MJ, McCleland ML, Stukenberg PT, Gorbsky GJ (2002b) Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. Curr Biol 12:900–905PubMedCrossRefGoogle Scholar
  28. Katayama H, Brinkley WR, Sen S (2003) The Aurora kinases: role in cell transformation and tumorigenesis. Cancer Metastasis Rev 22:451–464PubMedCrossRefGoogle Scholar
  29. Klein UR, Nigg EA, Gruneberg U (2006) Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of borealin, survivin, and the N-terminal domain of INCENP. Mol Biol Cell 17:2547–2558PubMedCrossRefGoogle Scholar
  30. Kwon M, Scholey JM (2004) Spindle mechanics and dynamics during mitosis in Drosophila. Trends Cell Biol 14:194–205PubMedCrossRefGoogle Scholar
  31. Kwon M, Morales-Mulia S, Brust-Mascher I, Rogers GC, Sharp DJ, Scholey JM (2004) The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility. Mol Biol Cell 15:219–233PubMedCrossRefGoogle Scholar
  32. Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt GA, Shabanowitz J, Hunt DF, Walczak CE, Stukenberg PT (2004) Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity. Curr Biol 14:273–286PubMedGoogle Scholar
  33. Lens SM, Wolthuis RM, Klompmaker R, Kauw J, Agami R, Brummelkamp T, Kops G, Medema RH (2003) Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension. Embo J 22:2934–2947PubMedCrossRefGoogle Scholar
  34. Mackay AM, Ainsztein AM, Eckley DM, Earnshaw WC (1998) A dominant mutant of inner centromere protein (INCENP), a chromosomal protein, disrupts prometaphase congression and cytokinesis. J Cell Biol 140:991–1002PubMedCrossRefGoogle Scholar
  35. Miyauchi K, Zhu X, Foong C, Hosoya H, Murata-Hori M (2007) Aurora B kinase activity is required to prevent polar cortical ingression during cytokinesis. Cell Cycle 6:2549–2553PubMedGoogle Scholar
  36. Murata-Hori M, Wang YL (2002) Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B. J Cell Biol 159:45–53PubMedCrossRefGoogle Scholar
  37. Murata-Hori M, Tatsuka M, Wang YL (2002) Probing the dynamics and functions of aurora B kinase in living cells during mitosis and cytokinesis. Mol Biol Cell 13:1099–1108PubMedCrossRefGoogle Scholar
  38. Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8:379–393PubMedCrossRefGoogle Scholar
  39. 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 8:78–83PubMedCrossRefGoogle Scholar
  40. Rosasco-Nitcher SE, Lan W, Khorasanizadeh S, Stukenberg PT (2008) Centromeric Aurora-B activation requires TD-60, microtubules, and substrate priming phosphorylation. Science 319:469–472PubMedCrossRefGoogle Scholar
  41. Ruchaud S, Carmena M, Earnshaw WC (2007) Chromosomal passengers: conducting cell division. Nat Rev Mol Cell Biol 8:798–812PubMedCrossRefGoogle Scholar
  42. Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT, Musacchio A (2005) Mechanism of Aurora B activation by INCENP and inhibition by hesperadin. Mol Cell 18:379–391PubMedCrossRefGoogle Scholar
  43. Shah JV, Botvinick E, Bonday Z, Furnari F, Berns M, Cleveland DW (2004) Dynamics of centromere and kinetochore proteins; implications for checkpoint signaling and silencing. Curr Biol 14:942–952PubMedGoogle Scholar
  44. Sharp DJ, Rogers GC (2004) A Kin I-dependent Pacman-flux mechanism for anaphase A. Cell Cycle 3:707–710PubMedGoogle Scholar
  45. Sharp DJ, Brown HM, Kwon M, Rogers GC, Holland G, Scholey JM (2000) Functional coordination of three mitotic motors in Drosophila embryos. Mol Biol Cell 11:241–253PubMedGoogle Scholar
  46. Stukenberg PT, Burke DJ (2004) Analyzing the spindle checkpoint in yeast and frogs. Methods Mol Biol 280:83–98PubMedGoogle Scholar
  47. Wheatley SP, Carvalho A, Vagnarelli P, Earnshaw WC (2001) INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis. Curr Biol 11:886–890PubMedCrossRefGoogle Scholar
  48. Yuen KW, Montpetit B, Hieter P (2005) The kinetochore and cancer: what’s the connection? Curr Opin Cell Biol 17:576–582PubMedCrossRefGoogle Scholar
  49. Zhang X, Lan W, Ems-McClung SC, Stukenberg PT, Walczak CE (2007) Aurora B Phosphorylates Multiple Sites on Mitotic Centromere-associated Kinesin to Spatially and Temporally Regulate Its Function. Mol Biol Cell 18:3264–3276PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Leena J. Ahonen
    • 1
    • 2
  • Anu M. Kukkonen
    • 1
  • Jeroen Pouwels
    • 1
  • Margaret A. Bolton
    • 3
  • Christopher D. Jingle
    • 3
  • P. Todd Stukenberg
    • 3
  • Marko J. Kallio
    • 1
    • 4
  1. 1.VTT Technical Research Centre of Finland, Medical BiotechnologyUniversity of TurkuTurkuFinland
  2. 2.Turku Graduate School of Biomedical SciencesTurkuFinland
  3. 3.Department of Biochemistry and Molecular GeneticsUniversity of Virginia Medical SchoolCharlottesvilleUSA
  4. 4.TurkuFinland

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