Figure 1.
Figure 1. Characterization of a novel nuclear shrinking assay. (A) In vivo: diagrams of X. laevis embryos are reprinted from Nieuwkoop and Faber (1956), and images of NPC-stained endogenous embryonic nuclei are adapted from Levy and Heald (2010; with permission from Elsevier). In vitro: nuclei assembled in X. laevis egg extract were incubated in LEE and visualized by NPC staining (mAb414). Complete details of the assay are described in the Materials and methods section. (B) Confocal z stacks (3-µm-thick sections) were acquired and maximum intensity projections are shown for representative nuclei. The control nuclei were treated with HI-LEE. n > 10 nuclei and 3 different extracts. (C) 3D surface plots are shown for the nuclei in B. (D) Nuclei treated with HI-LEE (control) and LEE were stained with mAb414. Nuclear surface area was calculated directly from confocal z stacks (blue bars), and nuclear surface area was then estimated for those same nuclei by measuring the cross-sectional area and multiplying by four (green bars). These values agreed within 3% (P > 0.7), which is consistent with these nuclei having roughly spherical geometry and validating our approach of estimating total NE surface area from the cross-sectional area. n = 20 nuclei each, error bars represent SD. (E) Nuclear shrinking data from 46 different extracts are shown. “Control Treated Nuclei” represent nuclei incubated in either extract buffer or HI-LEE. Each bar shows the mean for >240 nuclei. Error bars represent SD. (F) Nuclei were assembled de novo in X. laevis egg extract supplemented with recombinant GFP-LB3 and incubated in LEE. Live time-lapse imaging was performed at 30-s intervals for 90 min (see Video 1). Figure panels show 10-min intervals of a representative shrinking nucleus. (G) De novo assembled nuclei were incubated in LEE or HI-LEE, fixed at 30-min intervals, and quantified. Error bars represent SD. One representative experiment out of eight is shown. (H) Box-and-whisker plots are shown comparing fold changes in nuclear surface area. The blue (control nuclei) and green (LEE-treated nuclei) bars show in vitro data from one representative experiment (n > 200 nuclei for each treatment), normalized to the mean size for the control treated nuclei. The purple (stage 10) and red (stage 14) bars are in vivo nuclear sizes (n > 140 nuclei for each stage), normalized to the mean size for stage 10 nuclei. Boxes represent 50% of the data and the thick black line represents the median. The upper and lower error bars represent the upper and lower quartile of the data, respectively. Bars: (A) 20 µm; (B and C) 5 µm; (F) 10 µm.

Characterization of a novel nuclear shrinking assay. (A) In vivo: diagrams of X. laevis embryos are reprinted from Nieuwkoop and Faber (1956), and images of NPC-stained endogenous embryonic nuclei are adapted from Levy and Heald (2010; with permission from Elsevier). In vitro: nuclei assembled in X. laevis egg extract were incubated in LEE and visualized by NPC staining (mAb414). Complete details of the assay are described in the Materials and methods section. (B) Confocal z stacks (3-µm-thick sections) were acquired and maximum intensity projections are shown for representative nuclei. The control nuclei were treated with HI-LEE. n > 10 nuclei and 3 different extracts. (C) 3D surface plots are shown for the nuclei in B. (D) Nuclei treated with HI-LEE (control) and LEE were stained with mAb414. Nuclear surface area was calculated directly from confocal z stacks (blue bars), and nuclear surface area was then estimated for those same nuclei by measuring the cross-sectional area and multiplying by four (green bars). These values agreed within 3% (P > 0.7), which is consistent with these nuclei having roughly spherical geometry and validating our approach of estimating total NE surface area from the cross-sectional area. n = 20 nuclei each, error bars represent SD. (E) Nuclear shrinking data from 46 different extracts are shown. “Control Treated Nuclei” represent nuclei incubated in either extract buffer or HI-LEE. Each bar shows the mean for >240 nuclei. Error bars represent SD. (F) Nuclei were assembled de novo in X. laevis egg extract supplemented with recombinant GFP-LB3 and incubated in LEE. Live time-lapse imaging was performed at 30-s intervals for 90 min (see Video 1). Figure panels show 10-min intervals of a representative shrinking nucleus. (G) De novo assembled nuclei were incubated in LEE or HI-LEE, fixed at 30-min intervals, and quantified. Error bars represent SD. One representative experiment out of eight is shown. (H) Box-and-whisker plots are shown comparing fold changes in nuclear surface area. The blue (control nuclei) and green (LEE-treated nuclei) bars show in vitro data from one representative experiment (n > 200 nuclei for each treatment), normalized to the mean size for the control treated nuclei. The purple (stage 10) and red (stage 14) bars are in vivo nuclear sizes (n > 140 nuclei for each stage), normalized to the mean size for stage 10 nuclei. Boxes represent 50% of the data and the thick black line represents the median. The upper and lower error bars represent the upper and lower quartile of the data, respectively. Bars: (A) 20 µm; (B and C) 5 µm; (F) 10 µm.

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