Stress-specific differences in assembly and composition of stress granules and related foci - PubMed (original) (raw)

. 2017 Mar 1;130(5):927-937.

doi: 10.1242/jcs.199240. Epub 2017 Jan 17.

Affiliations

Anaïs Aulas et al. J Cell Sci. 2017.

Abstract

Cells have developed different mechanisms to respond to stress, including the formation of cytoplasmic foci known as stress granules (SGs). SGs are dynamic and formed as a result of stress-induced inhibition of translation. Despite enormous interest in SGs due to their contribution to the pathogenesis of several human diseases, many aspects of SG formation are poorly understood. SGs induced by different stresses are generally assumed to be uniform, although some studies suggest that different SG subtypes and SG-like cytoplasmic foci exist. Here, we investigated the molecular mechanisms of SG assembly and characterized their composition when induced by various stresses. Our data revealed stress-specific differences in composition, assembly and dynamics of SGs and SG-like cytoplasmic foci. Using a set of genetically modified haploid human cells, we determined the molecular circuitry of stress-specific translation inhibition upstream of SG formation and its relation to cell survival. Finally, our studies characterize cytoplasmic stress-induced foci related to, but distinct from, canonical SGs, and also introduce haploid cells as a valuable resource to study RNA granules and translation control mechanisms.

Keywords: Haploid cell; Stress granules; Stress response; Translation initiation; Translational control; eIF2α.

© 2017. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.

Fig. 1.

HAP1 cells are suitable for cellular and in vitro translation assay. (A) HAP1, COS7, MEFs, HeLa-S and U2OS cells were subjected to RiboPuromycylation to compare levels of basal translation. An anti-puromycin antibody (Puro) was used to visualize de novo synthesized proteins. Actin is a loading control. A representative image is shown (_n_=3). (B) Schematic of NanoLuc-based luciferase mRNA reporters with cap structures (Cap-NanoLuc), a poly(A) tail (NanoLuc-A50), a cap and poly(A) (Cap-NanoLuc-A50) or without a 5′-cap or 3′-poly(A) tail (NanoLuc). (C) IVT system based on HAP1 lysates was used to assess 5′-cap or 3′-poly(A) tail synergy using in vitro transcribed reporters from B. Relative translation efficiency of NanoLuc mRNA is set as 1. _n_≥3. *P<0.05; ns, not significant compared with NanoLuc (unpaired Student's _t_-test). (D) Schematic of bicistronic constructs used. The first ORF encoding firefly luciferase is translated in cap-dependent manner, the second ORF encoding NanoLuc luciferase is translated in IRES-dependent (for Polio virus, EMCV or HCV IRESs) manner. Control reporter (no IRES) contains no IRES element between the ORF encoding firefly and NanoLuc luciferases. (E) IVT system based on HAP1 lysates was used to assess translation of in vitro transcribed bicistronic reporters compared with the relative translation efficiency of firefly luciferase (black columns) or NanoLuc luciferase (gray) mRNAs is set as 1, respectively. The relative translation of IRESs over No IRES is shown. _n_≥3; *P<0.05; unpaired Student's _t_-test. Quantitative results are mean±s.e.m.

Fig. 2.

Fig. 2.

Composition of stress-induced foci. (A) HAP1 cells were subjected to treatment with SA (200 µM, 1 h), heat shock (44°C, 1 h), the proteasome inhibitor MG132 (100 µM, 1 h), the endoplasmic reticulum stressor Thaps (4 µM, 2 h), the eIF4A inhibitors RocA (2 µM, 2 h) and pateamine A (PatA, 0.5 µM, 1 h), or were subjected to osmotic stress by treatment with NaCl (0.2 M, 1 h). Unstressed cells (Control) were used as a control. Cells were examined for the presence of he core SG marker G3BP1 (immunofluorescence using G3BP1-specific antibody) and poly(A) mRNAs [FISH using oligo(dT) probe]. G3BP1- or oligo(dT)-positive cells were quantified. Results are mean±s.e.m. (_n_=3). (B) Representative images of HAP1 cells stained with G3BP1 (green) and oligo(dT) (red) after the cells had been subjecting to the specific stresses. The boxed region is enlarged and line scans used to assess colocalization (separate colors shown on graphics) of markers. (C) Representative images of HAP1 cells stained with G3BP1 (green), eIF4G (red) and eIF3B (blue) after the cells had been subjected to the specific stresses or left untreated (Control). The boxed region is enlarged and line scans used to assess colocalization (separate colors shown on graphics) of the markers.

Fig. 3.

Fig. 3.

Relationship between stresses, eIF2α phosphorylation, translation inhibition and stress foci formation. (A) WT or eIF2αS51A (S51A) HAP1 cells were subjected to treatment with SA (200 µM, 1 h), heat shock (44°C, 1 h), MG132 (100 µM, 1 h), Thaps (4 µM, 2 h), RocA (2 µM, 2 h), PatA (0.5 µM, 1 h) or NaCl (0.2 M, 1 h). Cells were pulsed with puromycin for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin (Puro), p-eIF2α, total eIF2α and actin. Representative images are shown (_n_≥3). (B) IVT assay of NanoLuc mRNA reporter based on cell lysates prepared from WT and S51A HAP1 cells that were treated with SA or were left untreated (Control). Relative luciferase units are shown. _n_=3. *_P_≤0.05 compared with SA-treated WT (unpaired Student's _t_-test). (C) WT and S51A HAP1 cells were assessed for G3BP1-positive foci by immunofluorescence using G3BP1. Percentage of cells with G3BP1-positive foci is shown. _n_≥3. Quantitative results are mean±s.e.m.

Fig. 4.

Fig. 4.

Translation-dependent dynamics of SGs and stress-induced foci. (A,B) WT (A) and S51A (B) HAP1 cells were treated with SA, heat shock, MG132, Thaps, UV, RocA, PatA or NaCl as previously described (Fig. 3C). 30 min before collection cells were treated with CHX (50 µg/ml) or puromycin (20 µg/ml). Cells were assessed by G3BP1 staining and plotted as percentage on the graphs according to color code: gray, stress only; blue, stress with CHX; red, stress with puromycin. Results are mean±s.e.m., _n_≥3. *_P_≤0.05 compared with stress alone (unpaired Student's _t_-test). (C) Representatives images of cells from B stained with G3BP1, eIF4G and TIA-1.

Fig. 5.

Fig. 5.

Determination of the eIF2αK activated in response to stresses. (A) WT, ΔHRI, ΔPKR, ΔPERK and ΔGCN2 mutant cells were exposed to SA, heat shock, Thaps, MG132, UV or NaCl as described in previous figures. Cells were pulsed with puromycin for 5 min before lysis. Whole-cell extract was analyzed by western blotting for puromycin, p-eIF2α, total eIF2α and actin, _n_≥3. (B) Cells positive for G3BP1 staining were analyzed as in Fig. 2B. Results are mean±s.e.m., _n_≥3. *_P_≤0.05 compared with WT (unpaired Student's _t_-test).

Fig. 6.

Fig. 6.

Stress-specific influences on cell death in WT and S51A HAP1 cells. WT or eIF2αS51A mutant cells were exposed to SA, heat shock, Thaps, MG132, UV, PatA, RocA or NaCl. After stress, the medium was changed and cell death was assessed 24 h later by Trypan Blue exclusion. Results are mean±s.e.m., _n_≥3. *_P_≤0.05 compared with WT (unpaired Student's _t_-test).

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