Figure 4.
Cdc28 is recruited to SGs and modulates SG dynamics. (A) Maximum projections of confocal images from cells expressing Cdc8-GFP and Pub1-mCherry after 30 min at 42°C in the absence of glucose (top). Scale bar, 2 µm. GFP and mCherry foci detected for quantification in a sample cell with the aid of BudJ are also shown at the bottom (scale bar, 1 µm). (B) Cdc8-GFP levels in Pub1-mCherry foci (n = 60), and vice versa, from cells as in A are plotted in relative units (r.u.) with point diameter corresponding to foci size. Slope (m) values are also shown. (C) Cdc8-GFP levels in Pub1-mCherry foci were measured in WT and whi8 cells stressed for 30 min at 42°C in the absence of glucose. Individual data (n = 125) and median ± Q values are plotted. Shown P value was obtained using a Mann–Whitney U test. (D) Immunoblot of input and αFLAG immunoprecipitation samples from cells expressing Cdc28-6FLAG and Pub1-3HA. (E) Venn diagram showing the overlapping of proteins that (1) were identified as SG components and (2) are putative phosphorylation targets of Cdc28. The indicated P value was obtained assuming completely independent allocations. (F) WT, cdc28-13, and oCLN3 (tetO2-CLN3) cells expressing Pub1-mCherry were stressed for 30 min at 42°C in the absence of glucose, and, once released at 37°C in the presence of glucose, Pub1-mCherry levels in foci were measured at different time points. Mean values (n > 30) and confidence limits for the mean (α = 0.05) are plotted. (G)GAL1p-CLN3 cln1,2 cells expressing Pub1-mCherry were grown in galactose and arrested in G1 by transfer to glucose for 2 h (Cdk OFF). Cells were then stressed for 30 min at 42°C in the absence of glucose, and, once released at 30°C in the presence of glucose to maintain the G1 arrest, Pub1-mCherry levels in foci were measured at different time points. As control, CLN3 cln1,2 cells (Cdk ON) were subjected to the same experimental conditions. Mean values (n > 30) and confidence limits for the mean (α = 0.05) are plotted. (H and I) WT (gray) and cdc28-13 (orange) cells expressing the Gal4-hER-VP16 transactivator and plasmid-borne SNF2-GFP (H) or GFP (I) from the GAL1p promoter were added estradiol and immediately treated for 30 min at 42°C in the absence of glucose. Once released at 30°C in the presence of glucose, estradiol was removed to limit further accumulation of the SNF2-GFP mRNA. Single-cell fluorescence levels at the indicated time points are plotted. Median values (n > 200) and confidence limits for the median (α = 0.05) are also shown. (J) WT (gray) and cdc28-13 (orange) cells with plasmids expressing the indicated GFP fusions from the GAL1p promoter were analyzed as in H. Single-cell fluorescence levels at the indicated time points after release from stress are plotted in relative units (r.u.). Median values (n > 200) and confidence limits for the median (α = 0.05) are also plotted. Shown P values were obtained using a Mann–Whitney U test. wt, wild type.

Cdc28 is recruited to SGs and modulates SG dynamics. (A) Maximum projections of confocal images from cells expressing Cdc8-GFP and Pub1-mCherry after 30 min at 42°C in the absence of glucose (top). Scale bar, 2 µm. GFP and mCherry foci detected for quantification in a sample cell with the aid of BudJ are also shown at the bottom (scale bar, 1 µm). (B) Cdc8-GFP levels in Pub1-mCherry foci (n = 60), and vice versa, from cells as in A are plotted in relative units (r.u.) with point diameter corresponding to foci size. Slope (m) values are also shown. (C) Cdc8-GFP levels in Pub1-mCherry foci were measured in WT and whi8 cells stressed for 30 min at 42°C in the absence of glucose. Individual data (n = 125) and median ± Q values are plotted. Shown P value was obtained using a Mann–Whitney U test. (D) Immunoblot of input and αFLAG immunoprecipitation samples from cells expressing Cdc28-6FLAG and Pub1-3HA. (E) Venn diagram showing the overlapping of proteins that (1) were identified as SG components and (2) are putative phosphorylation targets of Cdc28. The indicated P value was obtained assuming completely independent allocations. (F) WT, cdc28-13, and oCLN3 (tetO2-CLN3) cells expressing Pub1-mCherry were stressed for 30 min at 42°C in the absence of glucose, and, once released at 37°C in the presence of glucose, Pub1-mCherry levels in foci were measured at different time points. Mean values (n > 30) and confidence limits for the mean (α = 0.05) are plotted. (G)GAL1p-CLN3 cln1,2 cells expressing Pub1-mCherry were grown in galactose and arrested in G1 by transfer to glucose for 2 h (Cdk OFF). Cells were then stressed for 30 min at 42°C in the absence of glucose, and, once released at 30°C in the presence of glucose to maintain the G1 arrest, Pub1-mCherry levels in foci were measured at different time points. As control, CLN3 cln1,2 cells (Cdk ON) were subjected to the same experimental conditions. Mean values (n > 30) and confidence limits for the mean (α = 0.05) are plotted. (H and I) WT (gray) and cdc28-13 (orange) cells expressing the Gal4-hER-VP16 transactivator and plasmid-borne SNF2-GFP (H) or GFP (I) from the GAL1p promoter were added estradiol and immediately treated for 30 min at 42°C in the absence of glucose. Once released at 30°C in the presence of glucose, estradiol was removed to limit further accumulation of the SNF2-GFP mRNA. Single-cell fluorescence levels at the indicated time points are plotted. Median values (n > 200) and confidence limits for the median (α = 0.05) are also shown. (J) WT (gray) and cdc28-13 (orange) cells with plasmids expressing the indicated GFP fusions from the GAL1p promoter were analyzed as in H. Single-cell fluorescence levels at the indicated time points after release from stress are plotted in relative units (r.u.). Median values (n > 200) and confidence limits for the median (α = 0.05) are also plotted. Shown P values were obtained using a Mann–Whitney U test. wt, wild type.

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