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. 2019 Aug 2;10:3490. doi: 10.1038/s41467-019-11442-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — Localization of the ubiquitin ligase adaptor spatially controls degradation. a Representative time-lapse images of a HeLa cell transiently expressing a nuclear export signal-tagged ubiquitin ligase adaptor (NES-TIR1) and a Venus- and mAID-tagged lamin LMNA. 0.5 mM NAA was added at t = 0 to induce auxin-dependent degradation. During interphase, Venus-mAID-LMNA in the nuclear lamina is protected from ubiquitination by cytosolic NES-TIR1 and fluorescence persists in the presence of NAA. Scale bar: 30 µm. See also Supplementary Movie 5. b Model of mAID-LMNA protection from cytosolic NES-TIR1 during interphase. c The same cell is shown entering mitosis, which is visible by cell rounding in the brightfield (BF) overlay. After nuclear envelope breakdown (NEBD, t = 0), Venus-mAID-LMNA is accessible by cytosolic NES-TIR1 and fluorescence rapidly disappears. See also Supplementary Movie 6. d mAID-tagged reporters in the nucleus are accessible to cytosolic NES-TIR1 after NEBD occurs during mitosis. e Interphase HeLa cell stably expressing a nuclear localization signal-tagged TIR1 (NLS-TIR1) and NES-TIR1 from a single mRNA in addition to Venus-mAID-LMNA. After treatment with 0.5 mM NAA (t = 0), Venus-mAID-LMNA fluorescence rapidly disappears during interphase. See also Supplementary Movie 7. f Model of mAID-LMNA accessibility to nuclear NLS-TIR1 during interphase. g Quantification of Venus-mAID-LMNA fluorescence before and after NEBD (t = 0) in the presence of cytosolic NES-TIR1 and NAA. Values were normalized to NEBD. h Quantification of Venus-mAID-LMNA fluorescence in interphase cells in the presence of cytosolic NES-TIR1, nuclear NLS-TIR1, and NAA. Values were normalized to t = 0 (NAA addition). i Comparison of Venus-mAID-LMNA degradation velocities from single cells. The degradation velocity was determined for 45 min before NEBD (interphase) and 45 min after NEBD (mitosis) for NES-TIR1-expressing cells from three independent experiments or during the first 45 min after NAA addition for cells expressing both NLS-TIR1 and NES-TIR1 from two independent experiments. Bars indicate the mean of the indicated number of cells. Significance according to unpaired multi-comparison Kruskal–Wallis test with Dunn’s statistical hypothesis testing (****p < 0.0001; ns, p > 0.9999). Source data are provided as a Source Data file