Fig. 6: Ubiquitination of Aurora B at Lys202 is required for proper mitotic progression. | Communications Biology

Fig. 6: Ubiquitination of Aurora B at Lys202 is required for proper mitotic progression.

From: The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis

Fig. 6

a Representative live-cell still images of dividing mCherry-H2B HeLa cells co-transfected with siAurora B and pEGFP-vector control, pEGFP-Aurora B or pEGFP-Aurora B-K202R, respectively. Bars, 5 µm. NEBD indicates the first frame after nuclear envelope breakdown, based on the chromatin marker mCherry-H2B. Times are shown in hours: min. mCherry-H2B HeLa cells transfected with the indicated plasmids were synchronized by RO-3306 (9 μM) for16 h and then released into a fresh medium. Live-cell confocal time-lapse images were taken at the indicated time points. b Quantification of the mitotic duration cells spent from NEBD to mitosis completion. **p < 0.01 c Quantification of the time course cells spent from NEBD to anaphase onset. **p < 0.01. Each dot represents an individual cell. d Representative images of multi-nuclei defects in post-mitotic cells co-transfected with siAurora B and pEGFP-vector control, pEGFP-Aurora B, pEGFP-Aurora B-K202R or pEGFP-Aurora B-K106R, a known kinase inactive mutant. Bar, 5 μm. e Quantification of the cells with multi-nuclei defects shown in d. Data are shown as means ± SD, n = 3 biological replicates. In each independent experiment, 30 cells were analyzed. f–i The effects of Aurora B-K202R mutant on KT-MT error correction. Representative confocal images showing metaphase chromosome alignments (f) or anaphase chromosomes segregation (h) in cells transfected with the indicated plasmids as described in d. HeLa cells were synchronized by monastrol (50 μM) and released into a fresh medium for 30 or 45 min. Bar, 5 μm. g Quantification of the cells with aligned chromosomes at metaphase shown in f. Data are shown as means ± SD, n = 3 biological replicates. In each independent experiment, 20 cells were analyzed. i Quantification of the cells with chromosome segregation defects at anaphase, shown in h. Data are shown as means ± SD, n = 3 biological replicates. In each independent experiment, 16 cells were analyzed. **p < 0.01 versus CT, Student’s t-test.

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