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. 2015 Jun 15;26(12):2227–2241. doi: 10.1091/mbc.E15-01-0026

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© 2015 Karg, Warecki, and Sullivan. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/bync-sa/3.0).

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FIGURE 6: — Local inhibition of nuclear envelope assembly is mediated by Aurora B kinase. (A) Fixed images of telophase nuclei from I-CreI–expressing control and I-CreI with Aurora B depleted–neuroblasts bearing I-CreI–induced acentrics stained with anti-lamin (green) and DAPI (red). Acentrics induced in I-CreI–expressing controls show little to no association with lamin B and form a nuclear envelope gap (green arrow). In contrast, acentrics in neuroblasts with reduced Aurora B result in both the absence of a nuclear envelope gap and clear associations with lamins on acentrics (green arrowheads). (B) I-CreI–induced acentrics in control neuroblasts resulted in nuclear envelope gaps in 79% (N = 13) of telophase nuclei. In contrast, acentrics in neuroblasts depleted of Aurora B resulted in a significant reduction (14%; p < 0.05; N = 25) in the formation of nuclear envelope gaps in telophase nuclei. (C) Acentrics in I-CreI–expressing control neuroblasts were coated with lamin B in 21% (N = 13) of telophase nuclei. In contrast, telophase nuclei of neuroblasts depleted of Aurora B resulted in an increase (41%; N = 25) of acentrics coated with lamins (not significant). (D) Time-lapse images of dividing neuroblasts from I-CreI–expressing controls (top) and I-CreI with Aurora B RNAi (bottom; Supplemental Video S5) expressing lamin B-GFP (green) and H2Av-RFP (red). I-CreI–induced acentrics are present in both. In I-CreI–expressing controls, acentrics (red arrows) enter telophase nuclei through nuclear envelope gaps (green arrow). In contrast, neuroblasts bearing acentrics with reduced Aurora B show no gaps. Consequently, acentrics (red arrows) in Aurora B–depleted neuroblasts remain outside of the nucleus and form micronuclei coated with lamin B (green arrowheads). (E) Bar graphs of micronuclei frequencies in I-CreI–expressing control, I-CreI with Aurora B RNAi, and Aurora B RNAi without I-CreI control neuroblasts. (F) Box plots showing the time elapsed from the onset of anaphase to the completion of NEF in neuroblasts expressing I-CreI and I-CreI with Aurora B RNAi. Early-segregating I-CreI–induced acentrics do not form gaps, whereas late-segregating I-CreI–induced acentrics do form gaps. Late-segregating acentrics in neuroblasts with reduced Aurora B expression fail to form gaps and complete NEF in a similar time frame to early-segregating wild-type acentrics. These results indicate that Aurora B mediates the local inhibition of NEF in neuroblasts. Asterisks indicate statistical significance. (G) Time-lapse images of dividing neuroblasts from I-CreI–expressing, dimethyl sulfoxide (DMSO)–treated controls (left) and I-CreI–expressing Binucleine-2–treated neuroblasts (right) expressing lamin B-GFP (green) and H2Av-RFP (red). In DMSO-treated controls, acentrics (red arrows) enter telophase nuclei through nuclear envelope gaps (green arrows). In contrast, Binucleine-2–treated neuroblasts show no gaps. Consequently, acentrics (red arrows) in Aurora B–depleted neuroblasts remain outside of the nucleus and form micronuclei coated with lamin B (green arrowhead).