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. 2019 Jun 3;8:e48186. doi: 10.7554/eLife.48186

Figure 4. Increased steady state nuclear compartmentalization in aging is mimicked in NPC assembly mutants.

(a) Heatmaps showing single-cell changes in localization (N/C ratios) of GFP (N = 49), GFP-NES (N = 75) and GFP-NLS (N = 66) reporter proteins during replicative aging. (b) N/C ratios of GFP-tcNLS, GFP-NES and GFP as the cells age. The line indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the data points, which are closest to 1.5 times above below the inter quartile range, data points above or below this region are plotted individually. Non-overlapping notches indicate that the samples are different with 95% confidence. The number of cells analyzed are GFP = 54, 51, 34; GFP-NLS = 74, 48, 57 and GFP-NES = 75, 41, 66 at time points 0 hr, 15 hr and 30 hr, respectively. (c) Heatmaps showing single-cell changes in localization (N/C ratios) of Nab2NLS-GFP (N = 53) and Pho4NLS-GFP (N = 56) reporter proteins during replicative aging. (d) Median N/C ratios of Nab2NLS-GFP and Pho4NLS-GFP as the cells age. The number of cells analyzed are Nab2NLS-GFP = 55, 52, 29 and Pho4NLS-GFP = 59, 58, 33 at time points 0 hr, 15 hr and 30 hr, respectively. (e) Deletion of apq12 increases nuclear compartmentalization of GFP-NLS and GFP-NES. The number of cells analyzed are GFP-NLS = 42, 48 and GFP-NES = 39, 34 for WT and Δapq12, respectively (f) Increased nuclear compartmentalization of GFP-NLS during early aging (10 hr of aging, median age of 2 divisions) in a Δvps4Δheh2 background. The number of cells analysed are 42 and 33, respectively. (g) Heatmap showing single-cell changes in localization (N/C ratios) of Srm1-GFP (N = 85) during replicative aging. (h) N/C ratios of Srm1-GFP increases as cells age. Numbers of cells analysed are N = 103, 125, 77 at time points 0 hr, 15 hr and 30 hr, respectively.

Figure 4.

Figure 4—figure supplement 1. Efflux rate constants in aging.

Figure 4—figure supplement 1.

(a), (b) Singe-cell measurements of the kinetics of loss of nuclear accumulation of GFP-NLS from young cell and cell with median replicative age 8. Time zero is the time point at which the red colored medium (ponceau red) containing Na-azide and 2-Deoxy-D-glucose reached the cells that are trapped in the device. The measurements are fitted to an exponential decay function and yield the efflux rate constant (kout). Only cells with p<0.05 and R2 > 0.2 (plotted in red) are represented in panel c; poor fits (blue lines) are excluded from the analysis. (c) Efflux rate constant of cells age 0 and cells age 8. The average Kout of old cells is lower than for young cells, but changes are not significant. Number of cells included in the analysis are Age 0–1 = 57 and 8 (Median) = 48.
Figure 4—figure supplement 2. The abundance of transport factors and NTR cargos does not change in aging.

Figure 4—figure supplement 2.

(a) Protein abundance of Crm1, Kap95, Kap60, Kap104 and Kap121 as measured in whole cell extracts of yeast cells of increasing replicative age. Data from Janssens et al. (2015). (b) (c) Localization of Crm1 (b) and Kap95 (c) during replicative aging to the nucleus relative to the cytosol (N/C ratio). The line indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually. Non-overlapping notches indicate that the samples are different with 95% confidence. The overall changes were thus not significant, although we note that based on a two-tailed Student’s T-test the N/C ratio for Kap95 is significantly increased after 15 hr (p=8.7×10−4). No significant correlation was found with age (Crm1: r = 0.15, p=0.09 and Kap95: r = 0.07, p=0.39), or lifespan (Crm1: r = 0.04, p=0.63 and Kap95: r = 0.11, p=0.16). Number of cells analysed at time points 0 hr, 15 hr, and 30 hr were for Kap95 = 155, 165, 72 and for Crm1 = 156, 138, 87. (d) Heatmap representation of changes in N/C ratio of Crm1-GFP (N = 134) and Kap95-GFP (N = 132). (e) Protein abundance of 507 proteins with the Gene Ontology term ‘nucleus’ as measured in whole cell extracts of yeast cells of increasing replicative age. Data from Janssens et al. (2015). This set of proteins provides an unbiased proxy of changes in total import. The median, average or summed abundance of these nuclear proteins does not change in aging. (f) Protein abundance of 13 known cargos of Kap60, Kap121, Kap104 and Crm1 and 17 additional proteins interacting with Kap121/Kap123 (based on Timney et al., 2006) as measured in whole cell extracts of yeast cells of increasing replicative age. Data from Janssens et al. (2015).
Figure 4—figure supplement 3. Replicative lifespan curves.

Figure 4—figure supplement 3.

Replicative lifespan curves of strains expressing reporter proteins, in comparison to BY4741; all grown on medium supplemented with raffinose and galactose. The overexpression of GFP alone did not result in any observable growth defect in young cells, but did impact the lifespan of the yeast cells. This impact on lifespan is likely related to a general stress resulting from the additional protein synthesis and is unlikely to be related to nuclear transport. To enable comparison of the three reporter proteins, GFP, GFP-NLS and GFP-NES, in aging, we tuned their expression such that the impact on lifespan was similar for all three. Total number of cells analysed per strain were GFP = 89, GFP-NLS = 96, GFP-NES = 75, BY4741 = 126.
Figure 4—figure supplement 4. Apq12 is an essential gene in BY4741, but not in W303.

Figure 4—figure supplement 4.

(a) PCR analysis of apq12 mutants confirming the genotype of the heterozygous diploid BY4743 apq12Δ/APQ12 and the haploid W303 apq12Δ. Strains were generated using the PCR toolbox (Janke et al., 2004) replacing the apq12 gene with a hygromycin resistance cassette (hphNT1) and the genotype was confirmed by PCR analysis using primers flanking the apq12 locus. We were not able to obtain transformants of haploid BY4741 with the hphNT1 in the apq12 locus. (b) Haploid W303 apq12Δ is sensitive to growth at 22°C, confirming the phenotype described by Scarcelli et al. (2007). (c) Tetrad dissection of spores generated from BY4743 apq12Δ/APQ12. Spores from the tetrads grew out in colonies with varying sizes, or often did not grow out at all. (d) Genotype of the resulting clones from the tetrad dissection was determined by PCR with primers flanking the apq12 locus, as described above for panel a). The clones that carry the hphNT1 in the apq12 locus also still carry a wildtype copy of APQ12 (clones 1a, 1 c, 2b and 3 c). The heterozygous diploid BY4743 apq12Δ/APQ12 shows genome instability, possibly resulting in an incorrect number of chromosomes after meiosis, enabling spores to carry a WT copy of APQ12 as well as the apq12 deletion.