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. 2012 Oct;23(19):3786-800.
doi: 10.1091/mbc.E12-04-0296. Epub 2012 Aug 8.

Translation suppression promotes stress granule formation and cell survival in response to cold shock

Sarah Hofmann  1 , Valeria Cherkasova, Peter Bankhead, Bernd Bukau, Georg Stoecklin
Affiliations

Translation suppression promotes stress granule formation and cell survival in response to cold shock

Sarah Hofmann et al. Mol Biol Cell. 2012 Oct.

Abstract

Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock-induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia.

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Figures

FIGURE 1:
FIGURE 1:
Cold shock induces SGs and represses translation in mammalian cells. (A) COS7 cells were grown at 37°C or incubated at 30, 20, or 10°C for 10 h or 4°C for 8 h. Subcellular localization of eIF3B was determined by IF staining, followed by wide-field fluorescence microscopy. (B) As positive control for SG formation, cells were treated with 500 μM Na arsenite for 1 h. (C) COS7 cells exposed to cold shock were stained by fluorescence in situ hybridization for poly(A) mRNA and counterstained for eIF3B by IF. (D–F) COS7 cells exposed to cold shock at 10°C for 10 h were stained for the SG markers eIF4G (D), eIF2α (E), and G3BP (F) in combination with eIF3B. (G) COS7 cells were transiently transfected with a vector encoding GFP-PABP, exposed to cold shock, and fixed. Localization of GFP-PABP was analyzed in relation to endogenous eIF3B. (H) To quantify SG formation over time, the relative number of cells containing SGs was determined. Average values ± SE from four experiments are shown. (I) COS7 cells were exposed to cold shock at 10°C or kept at 37°C for different time periods, as indicated. Newly synthesized proteins were labeled with [35S]methionine/cysteine. Cell lysates were separated on 5–20% polyacrylamide gels and stained with colloidal Coomassie (bottom) or visualized by autoradiography (top). (J) COS7 cells were labeled as in I, and translation rates were quantified by measuring incorporation of [35S]methionine/cysteine into precipitated protein using a scintillation counter. Values of counts per minute (cpm) normalized to the total amount of proteins are depicted in the graph. (K) Polysome profiles were recorded from COS7 cells either grown under control conditions at 37°C or subjected to cold shock at 10°C for up to 10 h. To determine the percentage of polysomal ribosomes, the area below the polysomal part of the curve was divided by the area below the subpolysomal and polysomal parts of the curve and represented as average ± SD in the bar graph.
FIGURE 2:
FIGURE 2:
Cold shock induces SGs in S. cerevisiae. (A) A genomically tagged yeast strain expressing Pub1-GFP was grown under control conditions at 30°C or incubated at 15 or 10°C for 4 h. Subcellular localization of Pub1-GFP was analyzed by confocal microscopy. (B) Yeast was either grown under control conditions at 30°C or exposed to cold shock at 0°C for 4 and 24 h. Localization of genomically tagged Pub1-GFP (green) and Pab1-mCherry (red) was analyzed by confocal microscopy. (C) Spheroplasts were prepared from a genomically tagged yeast strain expressing Pab1-myc after it was either grown under control conditions or exposed to cold shock at 0°C for 4 h. Poly(A) RNA (green) was localized by fluorescence in situ hybridization, and Pab1-myc (red) was visualized by IF staining. (D) The formation of SGs over time was quantified using automated image analysis. Two parameters were measured: the number of SGs per cell and optical section (yellow bars) and the percentage of Pub1-GFP signal in SGs (brown bars). Average values ± SE from three experiments are shown.
FIGURE 3:
FIGURE 3:
Rapid disassembly of SGs upon recovery from cold shock in S. cerevisiae. (A) Cells expressing Pub1 genomically tagged with GFP were exposed to cold shock at 0°C for 4 h and then returned to 30°C. Cells were fixed at 30-s intervals, and Pub1-GFP was visualized by confocal microscopy. Four optical sections were used for deconvolution, and the resulting maximum projection is depicted in the images. (B) The disassembly of yeast SGs over time was quantified using automated image analysis. Two parameters were measured: the number of SGs per cell and optical section (yellow bars) and the percentage of Pub1-GFP signal in SGs (brown bars). Average values ± SE from three experiments are shown. (C) Polysome profiles were recorded from yeast during recovery from cold shock. (D) The percentage of polysomal ribosomes was quantified as in Figure 1K, average ± SD.
FIGURE 4:
FIGURE 4:
Rapid disassembly of mammalian SGs upon recovery from cold shock. (A) SG disassembly was monitored in COS7 cells that were subjected to cold shock at 10ºC for 10 h and then returned to 37ºC for 5, 10, 15, and 20 min. SGs were visualized by fluorescence wide-field microscopy after IF staining for eIF3B. (B) To quantify SG disassembly over time, the relative number of COS7 cells containing SGs was determined. Average values ± SE from four experiments are shown. (C) Polysome profiles were recorded from COS7 cells that were grown under control conditions at 37°C or subjected to cold shock at 10°C for 10 h and then allowed to recover at 37°C for the indicated time points. In the bar graph, the percentage of polysomal ribosomes was quantified as in Figure 1K, average ± SD.
FIGURE 5:
FIGURE 5:
Cold shock induces PERK-dependent eIF2α phosphorylation. (A) COS7 cells were subjected to cold shock at 10°C for up to 12 h (lanes 1–7), and total protein lysates were analyzed by Western blotting for phospho(S51)-eIF2α and total eIF2α. α-Tubulin serves as loading control. As a positive control, COS7 cells were treated for 1 h with 500 μM Na arsenite (lane 8). (B) Wild-type (eIF2α-SS) MEFs were subjected to cold shock at 10°C (lanes 1–8) and analyzed by Western blotting as in A. For control, eIF2α-SS and eIF2α-AA MEFs were treated for 1 h with 500 μM Na arsenite (lanes 9 and 10). (C) COS7 cells were subjected to cold shock at 10°C for 10 h and returned to 37°C. During the recovery phase, phospho(S51)-eIF2α levels were monitored as in A. (D) Wild-type and PERK ko MEFs were subjected to cold shock (CS) at 10°C for 10 h (lanes 5 and 6) and analyzed by Western blotting as in A. As controls, cells were treated for 1 h with 500 μM Na arsenite (lanes 3 and 4) or 2 mM DTT (lanes 7 and 8). (E) Polysome profiles were recorded from wt and PERK ko MEFs. Cells were grown under control conditions, subjected to cold shock at 10°C for 10 h, or treated with 2 mM DTT for 1 h. Polysome profiles were recorded as in Figure 1K; the bar graph shows quantification of two independent experiments.
FIGURE 6:
FIGURE 6:
Cold shock–induced translation arrest and SG formation in the absence of phospho-eIF2α in mammalian cells. (A) Polysome profiles were recorded from eIF2α-SS and eIF2α-AA MEFs. Cells were grown under control conditions, treated with 500 μM Na arsenite for 1 h, or subjected to cold shock at 10°C for 10 h. Polysomal ribosomes were quantified as in Figure 1K; the bar graph shows average values ± SE from three independent experiments. (B) SG formation was monitored in eIF2α-SS and eIF2α-AA MEFs grown at 37ºC (control), subjected to cold shock for 10 h at 10ºC, or treated for 1 h with 500 μM Na arsenite. SGs were visualized by IF microscopy after staining for eIF3B. (C) The percentage of cells containing SGs was quantified; average values ± SE are based on three experiments.
FIGURE 7:
FIGURE 7:
Gcn2-independent translation arrest and SG formation upon cold shock in yeast. (A) Polysome profiles were recorded from wt S. cerevisiae and a gcn2Δ strain. Cells were grown under control conditions or subjected to cold shock at 0°C for 1 and 4 h. (B) Translation was quantified as in Figure 3D. (C) Subcellular localization of Pab1-GFP was analyzed by confocal microscopy in the gcn2Δ strain. (D) SGs were quantified in wt and gcn2Δ S. cerevisiae using automated image analysis, as in Figure 2D. Average values ± SE from three experiments are shown.
FIGURE 8:
FIGURE 8:
Cold shock causes mTOR inhibition. (A) Huh7 cells were grown under control conditions at 37°C, subjected to 10°C cold shock for 10 h, or treated with 0.2 μM rapamycin for 1 h. Cells were lysed, and the cytoplasmic fraction (input, lanes 1–3) was incubated with 7-methyl-GTP (cap) Sepharose. Proteins retained by cap-Sepharose (lanes 4–6) were visualized by Western blotting using antibodies against eIF4E, 4EBP1, eIF4AI, eIF3B, and PABP. (B) 4EBP1+2 double ko MEFs, as well as wt counterparts, were grown under control conditions or subjected to 10 h of cold shock at 10°C. Polysome profiles were recorded as in Figure 1K; the bar graph shows quantification of two independent experiments. (C) Huh 7 cells were exposed to cold shock at 10°C for 10 h or kept under control conditions at 37°C. Cell lysates were loaded onto 17.5–50% sucrose gradients and separated by ultracentrifugation. After fractionation, protein extracts were resolved on 5–20% polyacrylamide gels. eIF4G, eIF4E, eIF4A, eIF3B, PABP, and rpL7 were detected by Western blotting.
FIGURE 9:
FIGURE 9:
Cold shock causes energy depletion and AMPK activation. (A) COS7 cells were grown under control conditions at 37°C, subjected to cold shock at 10°C for up to 8 h, or treated with FCCP (5 μM) for 1 h. ATP levels in the cellular lysates were measured using recombinant firefly luciferase and represented as percentage of control. Shown are average values ± SE from n = 7 (cold shock) or n = 4 (FCCP) independent experiments. (B) COS7 cells were grown under control conditions at 37°C or subjected to cold shock at 10°C for 10 h. Cells were labeled with Mitotracker Orange CM-H2TMRos 1 h before fixation and analyzed by fluorescence microscopy. (C) COS7 cells were grown under control conditions at 37°C or subjected to cold shock (CS) at 10°C for 9 h and then labeled for 1 h with Mitotracker Orange CM-H2TMRos at 10°C. The intensity of mitotracker staining was measured by flow cytometry. (D) COS7 cells were grown under control conditions at 37°C or subjected to cold shock at 10°C for up to 24 h. Total protein lysates were analyzed by Western blotting for phospho(T172)-AMPK, total AMPK, and eIF3A as loading control. (E) COS7 cells were subjected to cold shock at 10°C for 10 h and returned to 37°C. During the recovery phase, phospho(T172)-AMPK levels were monitored as in D.
FIGURE 10:
FIGURE 10:
AMPK and Scr kinase inhibitors attenuate SG formation, translation inhibition, and cell survival during cold shock. (A) COS7 cells were subjected to cold shock at 10°C for 4 or 8 h and simultaneously treated with 20 μM compound C. Cells were fixed and stained for eIF3B by IF. (B, C) Polysome profiles were recorded from COS7 cells subjected to cold shock at 10°C for (B) 4 h and (C) 8 h, in either the absence or presence of 20 μM compound C. (D) Translation was quantified in cold-shocked cells, in either the absence or presence of 20 μM compound C, by measuring the percentage of polysomal ribosomes as in Figure 1K. Average values ± SE from three experiments are shown. (E) COS7 cells were subjected to cold shock at 10°C for 8 h and simultaneously treated with 20 μM Src inhibitor-1. Cells were fixed and stained for eIF3B by IF. (F) Cell death was measured by the uptake of propidium iodide in unpermeabilized cells using flow cytometry. Before propidium iodide staining, COS7 cells were grown under control conditions at 37°C, treated for 10 h with 20 μM compound C or 20 μM Src inhibitor-1, or subjected to cold shock at 10°C for up to 10 h, in the absence or presence of 20 μM compound C or 20 μM Src inhibitor-1. The percentage of damaged or dead, that is, propidium iodide–positive cells is presented as average value ± SE, n = 5. (G) In addition to compound C (20 μM), cells were exposed to the translation inhibitor cycloheximide (CHX, 10 μg/ml) or puromycin (puro, 1 μg/ml) during the entire 10-h cold shock. Cell death was measured as described above; shown are average values ± SE, n = 6.
FIGURE 11:
FIGURE 11:
Mechanisms suppressing translation in mammalian cells exposed to cold shock. The model depicts parallel pathways that contribute to cold shock–induced translation suppression. Passive mechanisms include generally reduced enzymatic activities at lower temperatures and reduced mitochondrial function with consecutive drop in ATP levels. Active mechanisms include activation of AMPK, inhibition of TORC1, and PERK-dependent phosphorylation of eIF2α and may involve additional pathways. Translation suppression promotes cell survival under conditions of cold shock.
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