Figure 5. Inhibition of nuclear export rapidly leads to an increase in the N/C ratio and changes in crowding.

(A) Individual cells expressing plasma membrane and nuclear markers were imaged in time upon treatment with LMB or control (Ctrl). Images show a mid-focal plane of plasma membrane (green) and nuclear membrane (purple) treated with LMB (top) or not (Ctrl, bottom) over time (min). (B) Cell volumes were measured from 3D images. (C) Same as (B) for nuclear volume. (D) Box and whisker plot of N/C ratio of individual cells treated with LMB (cyan) and control condition (blue) at t=0 min and followed by time lapse microscopy. (E) N/C ratio dynamics of representative individual cells extracted from (D). (F) Cells expressing chromosomally tagged proteins that mark the large ribosomal subunit (Rpl3001-GFP) were treated with LMB and imaged over time. Mid focal plane confocal images and quantitation of their relative fluorescence intensities are displayed. Kruskal-Wallis statistical test was used. (G) Cells expressing cytoplasmic or nuclear GEMs were treated with LMB or control (0.05% ethanol) and were imaged for GEMs diffusion over time. Bar graphs show the relative changes in mean effective diffusion coefficients ± SEM for cytoplasmic (green) and nucleoplasmic (purple) GEMs in cells treated with LMB (left panel) and ethanol control (right panel). Statistical differences compared with Mann-Whitney U test. (A–G) From at least three biological replicates. (H) Cells were stained for total protein and RNA using FITC dye, plots indicate ratios of FITC intensities in nuclear and cytoplasmic regions over time after the addition of LMB. Statistical differences compared with Kruskal-Wallis test (p-value = 0.077), from at two biological replicates. (I) Normalized non-osmotic volume over time for cells and their nuclei in protoplasts treated with LMB. Scale bar = 5 µm. See also Figure 5—figure supplements 1 and 2.

Figure 5—figure supplement 1. Effects of LMB on the N/C ratio and ribosomal protein localization.

(A) Bright field mid-focal plane (top) and max Z-projection (bottom) images of the plasma membrane (green) and nuclear membrane (purple) of whole cells treated with LMB over time. Cells were selected to have approximately the same size. (B–C) Whole-cell volume and nuclear volume of distinct populations of cells treated with LMB and initial condition. (D) N/C ratio of a population of cells treated with LMB (light blue) and initial condition (blue). N≥463 per time point. (E–F) Cells expressing chromosomally tagged proteins that mark the large ribosomal subunit (Rpl2401-GFP) and the small ribosomal subunit (Rps2-GFP) were treated with LMB and imaged over time. Mid focal plane confocal images and quantitation of their relative fluorescence intensities are displayed. Statistical differences between 0 and 45 min were compared with Kruskal-Wallis test. (G) To reveal the distribution of total protein and RNA cells were fixed and stained with FITC dye. FITC fluorescence intensities along the normalized cell length were measured. (H) Same as (G), to reveal the distribution of total protein cells were also treated with RNAse. (G–H) FITC intensities in nuclear and cytoplasmic regions are defined by the signal in purple and green bar. Only a subpopulation of cells was plotted. (I) Evolution over time of the ratios of FITC intensities in nuclear and cytoplasmic regions after LMB treatment. Kruskal-Wallis statistical test was used, from at three biological replicates. Scale bar = 5 µm.

Figure 5—figure supplement 2. Effects of LMB and osmotic shifts on protoplasts.

(A–C) Time course of N/C ratio cellular volume and nuclear volume in individual control and LMB-treated protoplasts. (D) Z-sum projection image of the plasma membrane (green) and nuclear membrane (purple) of protoplasts over time treated with LMB (right) or not (Ctrl, left). Scale bar = 5 µm. (E) Scatter plot of cell size and nuclear size for protoplasts in isotonic condition treated with LMB for 15 min (YE +0.4 M sorbitol) and immediately following osmotic shock. Black line, N/C ratio of cells under isotonic condition (N/C=9%). Dashed line, N/C ratio of cells in isotonic condition before the addition of LMB (N/C_T=0). (F) Same as (E) for protoplasts treated with LMB for 60 min. (G–H) Effect of osmotic shifts on the relative volumes (V/V_iso, mean ±STD) protoplasts treated with LMB for 15 minutes (N=618, from at least two biological replicates). (I–J) Same as (G–H) for protoplasts treated with LMB for 60 minutes. (N=245, from at least two biological replicates).