Journal: iScience

Article Title: Proteasome inhibition induces microtubule-dependent changes in nuclear morphology

doi: 10.1016/j.isci.2024.111550

Figure Lengend Snippet: Proteasome inhibitor MG132 affects nuclear morphology and integrity (A–G) Xenopus laevis egg extract was supplemented with 500 μM MG132 or an equivalent volume of buffer (Control). We tested a range of MG132 concentrations and selected 500 μM because it induced the greatest effect on nuclear morphology ( Figure S4 ). GFP-NLS was added at 0.4 μg/μL for imaging, nuclear assembly was initiated as described in , and live widefield imaging was performed for 1 h at 30 s intervals. Once import-competent nuclei formed, quantification was performed for the subsequent 10 min to interrogate initial nuclear growth and import rates immediately after nuclear assembly. Data were acquired for 15 nuclei per condition and three biological replicates. (A-B) Images from representative time-lapses are shown. The scale bar is 20 μm. (C) At the indicated time points, nuclei were thresholded based on GFP-NLS signal and nuclear cross-sectional (CS) area was quantified and plotted as a function of time. (D) At the indicated time points, nuclei were thresholded and total nuclear GFP-NLS fluorescence intensity was quantified by multiplying average GFP-NLS pixel intensity by nuclear volume to obtain the integrated volumetric nuclear GFP-NLS signal (see ). These data were plotted as a function of time. (E) Nuclear growth rates were quantified based on the data shown in (C) by calculating the slope of the graph. (F) Nuclear import rates were quantified based on the data shown in (D) by calculating the slope of the graph. (G) When nuclei rupture, the intranuclear GFP-NLS signal disperses. To quantify the number of ruptured nuclei, we counted the number of nuclei positive for intranuclear GFP-NLS at the start of imaging and subtracted the number of nuclei still positive for intranuclear GFP-NLS after 1 h. This number was divided by the initial number of nuclei to obtain the rupture frequency. Ruptures typically occurred after the 10-min window used to calculate nuclear growth and import rates. (H–J) Nuclei were assembled in Xenopus laevis egg extract. After nuclear assembly, extracts were supplemented with 500 μM MG132 or an equivalent volume of buffer (Control) and incubated for 45 min. Nuclei were fixed and immunofluorescence against the nuclear pore complex (NPC) was performed with mAb414. (H) Representative widefield images are shown. The scale bar is 20 μm. (I) After thresholding, nuclear CS area was quantified and normalized to controls. (J) Thresholded images were quantified for nuclear roundness, which is the ratio of CS area to the square of the major axis. Nuclear CS area and roundness were quantified for >100 nuclei per condition and three biological replicates. (K–M) eGFP-LMNA HeLa cells were treated with 200 μM MG132 or an equivalent volume of buffer (Control) for 1 h and fixed. (K) Representative confocal images are shown. The scale bar is 20 μm. (L) After thresholding, nuclear CS area was quantified and normalized to controls. (M) Feret’s diameter, which is a measure of the longest distance between two points in a selected boundary, was calculated from thresholded images and used as a metric to quantify nuclear shape in HeLa cells. Nuclear CS area and Feret’s diameter were quantified for >100 nuclei per condition and three biological replicates. Error bars represent SD, except for in (D) where error bars are SEM. Unpaired two-tailed t-tests: ns, not significant; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S1 and .

Article Snippet: Widefield imaging was performed with an Olympus BX63 upright widefield epifluorescence microscope having multi-mode time-lapse imaging ability with an X-Cite 120 LED illumination system.

Techniques: Control, Imaging, Fluorescence, Incubation, Immunofluorescence, Two Tailed Test

Journal: iScience

Article Title: Proteasome inhibition induces microtubule-dependent changes in nuclear morphology

doi: 10.1016/j.isci.2024.111550

Figure Lengend Snippet: Microtubules contribute to the altered nuclear morphology induced by MG132 (A–D) Nuclei were assembled in Xenopus laevis egg extract. After nuclear assembly, extracts were supplemented with 500 μM MG132 and/or 33 μM nocodazole, as indicated. After a 45-min treatment, nuclei were fixed and immunofluorescence against the NPC was performed with mAb414. (A) Representative widefield images are shown. The scale bar is 20 μm. (B–C) Nuclear CS area and roundness were quantified as in Figure 1 . (D) Heterogeneity in NPC distribution, which reflects the wrinkled appearance of the nuclear envelope, was quantified by drawing line scans within the nuclear interior and calculating the standard deviation of the NPC signal intensity along the line. Nuclear CS area, roundness, and heterogeneity in NPC distribution were quantified for >100 nuclei per condition and three biological replicates. (E–G) eGFP-LMNA HeLa cells were treated with 200 μM MG132 and/or 3 μM nocodazole for 1 h and fixed. (E) Representative confocal images are shown. The scale bar is 20 μm. (F–G) Nuclear CS area and Feret’s diameter were quantified as in Figure 1 for >100 nuclei per condition and three biological replicates. Error bars represent SD. Ordinary One-Way ANOVA: ns, not significant; ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: Widefield imaging was performed with an Olympus BX63 upright widefield epifluorescence microscope having multi-mode time-lapse imaging ability with an X-Cite 120 LED illumination system.

Techniques: Immunofluorescence, Standard Deviation