Chromosoma

, 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

Abstract

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.

Notes

Acknowledgements

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)
412_2008_178_MOESM3_ESM.mov (8.1 mb)
Video 1 Xeno S3 cell injected with Incenp-ab at prophase (MOV 8.07 MB).
412_2008_178_MOESM4_ESM.mov (4.6 mb)
Video 2 Xeno S3 cell injected with Incenp-ab at prophase (MOV 4.61 MB).
412_2008_178_MOESM5_ESM.mov (619 kb)
Video 3 GFP-tubulin expressing Xeno S3 cell injected with Incenp-ab at metaphase (MOV 619 KB).
412_2008_178_MOESM6_ESM.mov (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).
412_2008_178_MOESM7_ESM.mov (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).
412_2008_178_MOESM8_ESM.mov (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).
412_2008_178_MOESM9_ESM.mov (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).
412_2008_178_MOESM10_ESM.mov (1.6 mb)
Video 8 Xeno S3 cell injected with Incenp-ab at the onset of anaphase (MOV 1.56 MB).
412_2008_178_MOESM11_ESM.mov (1.3 mb)
Video 9 Xeno S3 cell injected with Incenp-ab 1 min after the onset of anaphase (MOV 1.28 MB).
412_2008_178_MOESM12_ESM.mov (3.2 mb)
Video 10 Xeno S3 cell injected with Incenp-ab 3 min after the onset of anaphase (MOV 3.23 MB).
412_2008_178_MOESM13_ESM.mov (3.7 mb)
Video 11 Xeno S3 cell injected with Incenp-ab 5 min after the onset of anaphase (MOV 3.67 MB).
412_2008_178_MOESM14_ESM.mov (507 kb)
Video 12 GFP-tubulin expressing Xeno S3 cell injected with Incenp-ab at early anaphase (MOV 506 KB).
412_2008_178_MOESM15_ESM.mov (662 kb)
Video 13 GFP-tubulin expressing Xeno S3 cell injected with control buffer at early anaphase (MOV 662 KB).

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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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