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Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

Figure 1

Nrd1 localizes to stress granules under various environmental stresses.

(A) Analysis of Nrd1-GFP localization under stress. Localization of Nrd1-GFP in living cells grown at 27°C (untreated) after a shift to 42°C for 20 min (42°C 20 min) and after exposure to 2.0 mM arsenite (2.0 mM arsenite 120 min), 5.0 mM H2O2 (5.0 mM H2O2 30 min), 10.0 mM CdCl2 (10.0 mM CdCl2 120 min), or 1.0 M KCl (1.0 M KCl 10 min) for the times indicated. Wild-type (wt) cells transformed with pREP1-GFP-Nrd1 were grown in EMM (thiamine-free medium) for 18 h to induce overproduction of GFP-Nrd1 (overproduction 18 hr). Bar, 10 µm. The number in the picture indicates the SG number/cell in each experiment. Right panel: Quantitative analysis of the number of SGs/cell on each stress. Graph depicting the number of stress granules per cell formed before (untreated) and after each condition as indicated in Figure 1(A) plotted against time after exposure to each stress and the inset is a magnification of the results obtained on KCl treatment. (B) Co-localization of Nrd1 with poly(A)-binding protein (Pabp). Merged image of fluorescence micrographs showing Pabp-GFP (green) and Nrd1-tdTomato (red) in untreated cells and after a 20-min incubation at 42°C. Bar, 10 µm. (C) Fluorescence micrographs of the wild-type cells expressing mCherry-tagged Nrd1 grown at 26°C (untreated); and these were subjected to in situ hybridization with a digoxigenin-labeled oligo (dT)50 probe after a 40-min exposure to 2.0 mM arsenite (2.0 mM arsenite 40 min). The hybridized probe was detected by treatment with mouse anti-digoxin antibody, followed by a fluorescein-conjugated goat anti-mouse IgG antibody (FITC). Nrd1 was detected using mCherry fluorescence (mCherry-Nrd1). Nuclei are counterstained using DAPI dye (DAPI). Bar, 10 µm. (D) Cycloheximide (CHX) prevents the formation of heat-shock- and arsenite-induced Nrd1 granules. Fluorescent images of cells expressing Nrd1-GFP incubated (from left to right) at 27°C with 100 µg/ml CHX for 30 min (CHX); at 42°C for 20 min; pre-incubated with CHX for 30 min at 27°C followed by 20-min incubation at 42°C (CHX then 42°C 20 min); 20-min incubation at 42°C followed by CHX incubation (42°C 20 min then CHX); with 2.0 mM arsenite for 120 min at 27°C; pre-incubated with CHX for 30 min followed by 120-min incubation with 2.0 mM arsenite; and 120-min pre-incubation with arsenite followed by 30-min incubation at with CHX. Bar, 10 µm. Lower panel: Graph depicting the number of stress granules per cell in each condition plotted against time after exposure to each stress.

Figure 1

doi: https://doi.org/10.1371/journal.pone.0029683.g001