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.
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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.
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Communicated by: E. A. Nigg
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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. (d–e) 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)
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Video 4 Recovery of photobleached GFP-xIncenp at the centromeres of a Xeno S3 cell in the presence of MG132 (MOV 1.36 MB).
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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).
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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).
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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).
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Ahonen, L.J., Kukkonen, A.M., Pouwels, J. et al. Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres. Chromosoma 118, 71–84 (2009). https://doi.org/10.1007/s00412-008-0178-0
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DOI: https://doi.org/10.1007/s00412-008-0178-0