Yeast strains and culture
strains used in this study are listed in Supplemental Table S1. Standard yeast genetic techniques were applied throughout this study. Genomic tagging with mCherry, EGFP, and myc was carried out as described (Janke et al., 2004
), and transformations were performed using the lithium acetate method (Schiestl and Gietz, 1989
). For all experiments, cells were grown to OD600
0.4–0.6 in synthetic complete dextrose (SCD) medium containing 2% glucose at 30°C. To induce cold shock, cells were harvested by centrifugation, resuspended in fresh, ice-cold medium, and incubated on ice with continuous shaking for different time periods. For recovery experiments, cells were directly transferred from ice to a shaking water bath prewarmed to 30°C. For glucose depletion experiments, cells were grown to exponential phase, filtered using a vacuum pump, washed, and resuspended in SC medium lacking glucose, and incubated at 30°C for 10 min. For recovery experiments, glucose was added to starved cells to a final concentration of 2%.
Mammalian cell culture
HeLa, Huh7, and COS7 cells, as well as MEFs, were cultured in DMEM containing 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria), 2 mM l
-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all PAN Biotech, Aidenbach, Germany) at 37°C in 5% CO2
. DU145 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, l
-glutamine, penicillin, and streptomycin. eIF2α-SS and eIF2α-AA MEFs (Scheuner et al., 2001
) were a kind gift from R. Kaufman (University of Michigan Medical Center, Ann Arbor, MI). 4EBP1+2 ko MEFs (Dowling et al., 2010
) were kindly provided by Nahum Sonenberg (McGill University, Montreal, Canada), AMPKα1+2 ko MEFs (Laderoute et al., 2006
) were a kind gift from Benoit Viollet (Institut Cochin, Université Paris Descartes, Paris, France), and PERK ko MEFs (Harding et al., 2003
) were kindly provided by David Ron (New York University School of Medicine, New York, NY). Plasmid pSRα-HA-GFP-PABP (p2024) was described previously (Kedersha et al., 2000
) and was transfected into COS7 cells using polyethyleneimine (1 mg/ml, pH 7.0; Polysciences Europe, Eppelheim, Germany). Cells were replated onto glass coverslips 12 h before cold shock.
Mammalian cells were grown on glass coverslips and fixed at 4ºC in 4% paraformaldehyde for 10 min before cell membranes were permeabilized in –20°C cold methanol for 10 min. Phosphate-buffered saline (PBS) containing 0.1% sodium azide and 5% horse serum was used for blocking and antibody dilution. Cy3- or Cy2-conjugated secondary donkey antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for detection of primary antibodies. Cells were mounted onto glass slides using a solution of 14% polyvinyl alcohol (P8136; Sigma-Aldrich, St. Louis, MO) and 30% glycerol in PBS. SGs were counted on an upright epifluorescence microscope (BX60; Olympus, Tokyo, Japan). Images were acquired at the Nikon Imaging Center, Heidelberg, on an upright epifluorescence microscope (90i; Nikon, Melville, NY) using an electron-multiplying charge-coupled device (EM-CCD) camera (Hamamatsu, Hamamatsu, Japan) and at the Imaging Facility of the Zentrum für Molekulare Biologie der Universität Heidelberg using the Olympus Xcellence Pointfrap microscope and a Hamamatsu Orca-R2 camera.
Yeast cells were grown at 30°C, centrifuged at room temperature, resuspended in ice-cold SCD, and incubated in ice water for the indicated time periods. Cells were fixed with 4% paraformaldehyde and washed three times before microscopy. To visualize the entire cell volume, optical sections at a distance of 0.2 μm were acquired on a spinning disk confocal microscope (UltraView ERS [PerkinElmer, Waltham, MA] on a TE2000 inverted microscope [Nikon]) equipped with an EM-CCD camera (Hamamatsu) at the Nikon Imaging Center, Heidelberg. The entire stack was used for deconvolution using Huygens software (Scientific Volume Imaging, Hilversum, Netherlands). A maximum-intensity projection of three optical sections was generated for image representation, and contrast was enhanced using ImageJ software (National Institutes of Health, Bethesda, MD). For quantitative analysis, single-plane images were acquired and normalized to the exposure time.
Automated image analysis
An algorithm to calculate cell number, cell area, signal intensity within the cell, number of aggregates, area of aggregates, and signal intensity within aggregates from micrographs of S. cerevisiae was written in Matlab software (MathWorks, Natick, MA). Images were first normalized according to exposure times before cell and nuclear boundaries were determined. For the detection of aggregates in the cytoplasm, the local baseline fluorescence for each pixel within cells was calculated using opening by reconstruction, and aggregates were defined as pixels at which the fluorescence increase is >0.75 times the local baseline. The algorithm is available upon request.
Fluorescence in situ hybridization
Yeast spheroplasts were prepared as described previously (Finger et al., 1993
), spotted onto poly-l
-lysine–coated glass slides, and stored in 70% ethanol at −20°C. Acetylation was performed according to Schwab et al. (1998
). Briefly, the cells were washed once with 0.1 M triethanolamine, pH 8.0, incubated in 0.1 M triethanolamine/0.25% acetic anhydride for 10 min, and washed with PBS and 2× SSC. Hybridization with Alexa 488–conjugated oligo(dT)50
(Invitrogen, Carlsbad, CA) was performed overnight at 37°C in hybridization buffer containing 20% formamide, 2× SSC, 0.1% Triton X-100, 2% blocking reagent (Roche, Indianapolis, IN), and 10% dextran sulfate. After sequential washes with 2× SSC, 0.2× SSC, and PBS, samples were incubated for 1 h at room temperature in blocking buffer containing 1% blocking solution and 0.1% Triton X-100 in PBS. Cells were incubated with α-myc antibody (9E10, 1:500; Roche) overnight at 4°C in blocking buffer and further stained with 2 μg/ml Hoechst 33422 dye and Cy3-labeled secondary antibody diluted 1:500 in blocking buffer for 1 h at room temperature. Finally, cells were mounted in PBS containing 12.5% polyvinyl alcohol (Sigma-Aldrich), 25% glycerol, and 0.25% sodium azide.
Mammalian cells were fixed as described for IF, washed in 2× SSC, and incubated in a 1:5000 dilution of Alexa 555–coupled oligo(dT)50 probe (100 pmol/μl; Invitrogen) in hybridization buffer (1 mg/ml yeast RNA, 20% formamide, 2 mg/ml BSA, 0.1 g/ml dextran sulfate, 1× SSC) for 1 h at room temperature. Cells were then washed three times in 2× SSC at room temperature before mounting.
Cells were subjected to cold shock at 10°C for 10 h or kept under control conditions. At 1 h before the end of the treatment, 0.1 μM Mitotracker Orange CM-H2TMRos (Molecular Probes, Eugene, OR) was added to the cell culture. Cells were then collected by trypsinization and centrifugation, resuspended in PBS containing 2% fetal bovine serum, and analyzed on a FACSCanto II (BD Biosciences, Heidelberg, Germany) flow cytometer using Flowjo software (Tree Star, Ashland, OR). For microscopy, cells were grown on glass coverslips, stained with mitotracker as described, and fixed with 4% paraformaldehyde and −20°C methanol. Images were taken with a Leica DM5000B epifluorescence microscope (Leica, Wetzlar, Germany) and a CCD camera (Andor Technology, South Windsor, CT).
Western blot analysis
Cells were lysed in SDS sample buffer with 100 mM DTT. Proteins were resolved on 5–20% polyacrylamide gradient Tris-glycine gels and transferred onto nitrocellulose membranes of 0.2 μm pore size (Peqlab, Erlangen, Germany) for Western blotting. Membranes were then blocked in 5% horse serum in PBS containing 0.1% sodium azide, incubated with antibodies diluted in the same solution, and washed in 150 mM NaCl, 50 mM Tris, pH 7.5, and 1% Tween-20. Horseradish peroxidase–coupled secondary antibodies (Jackson ImmunoResearch Laboratories) in combination with Western Lightning enhanced chemiluminescence substrate (PerkinElmer) were used for detection.
The following antibodies were used for Western blot and IF: mouse monoclonal antibodies against G3BP1 (TT-Y, sc-81940; Santa Cruz Biotechnology, Santa Cruz, CA) and eIF2α (ab5369; Abcam, Cambridge, MA); polyclonal rabbit antibodies against phospho(S51)-eIF2α (KAP-CP131; Stressgen, San Diego, CA), eIF4GI (2498; Cell Signaling Technology, Frankfurt am Main, Germany), monoclonal rabbit phospho(T172)-AMPK (40H9, #2535; Cell Signaling Technology), phospho(S792)-raptor (#2083; Cell Signaling Technology); monoclonal rabbit antibodies against AMPKα (23A3, #2603; Cell Signaling Technology), 4EBP1 (53H11, #9644; Cell Signaling Technology), phospho(T37/46)-4EBP1 (#2855; Cell Signaling Technology), and raptor (#2280; Cell Signaling Technology); polyclonal goat antibody against eIF3B (sc-16377; Santa Cruz Biotechnology); and rat monoclonal antibody against α-tubulin (ab6160; Abcam).
Mammalian cells were treated with Na arsenite (500 μM, 1 h), rapamycin (0.2 μM, 1 h), or DTT (2 mM, 1 h) or were exposed to cold shock in either the absence or presence of compound C (20 μM) for the times indicated. Before lysis, 100 μg/ml cycloheximide (Sigma-Aldrich) was added to cells for 5 min, and cells were washed with cold PBS containing 100 μg/ml cycloheximide. Cells were harvested by scraping and lysed in 0.2 ml lysis buffer containing 15 mM Tris, pH 7.4, 15 mM MgCl2, 300 mM NaCl, 1% Triton X-100, 100 μg/ml cycloheximide, 500 μg/ml heparin, 0.2 U/ml RNasin (Promega, Madison, WI), 0.1% 2-mercaptoethanol, and EDTA-free protease inhibitor (Roche). Lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4°C. Supernatants were loaded onto linear gradients of 17.5–50% sucrose in 15 mM Tris, pH 7.4, 15 mM MgCl2, and 300 mM NaCl and centrifuged in a SW60 rotor at 35,000 rpm for 2.5 h at 4°C. Fractions were eluted from the top of the gradient using a Teledyne Isco (Lincoln, NE) gradient elution system; polysome profiles were obtained by measuring absorbance at 254 nm.
Yeast cultures were treated with 100 μg/ml cycloheximide on ice for 5 min, collected by centrifugation, washed once with ice-cold dH2O and resuspended in ice-cold yeast polysome lysis buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 50 mM KCl, 10 mM MgCl2, 300 mM NaCl, 1% TritonX-100, protease inhibitor cocktail [Roche], 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 100 μg/ml cycloheximide, and 50 U/ml Superasein [Ambion, Austin, TX]). Cell suspensions were then frozen in drops in liquid nitrogen and pulverized by mixer milling (MM 400; Retsch, Newtown, PA). Lysates were clarified by centrifugation at 14,000 × g for 10 min, and the amount corresponding to 1 mg of total RNA was loaded on 7–47% sucrose gradients. Gradients were centrifuged in a SW40 rotor at 35,000 rpm for 2.5 h and processed as described for mammalian cells. For quantification, “empty” gradients loaded with buffer alone were recorded using the same settings. Normalization was carried out by subtracting “empty gradient” values from “sample gradient” values, and the areas under the curve corresponding to the amount of polysomal and total ribosomes were determined by integration. Polysomal ribosomes were divided by total ribosomes as a measure for the translation rate.
Huh7 cells were kept under control conditions at 37°C or exposed to cold shock at 10°C for 10 h. Before lysis, cells were treated with 100 μg/ml cycloheximide for 5 min, washed with cold PBS containing 100 μg/ml cycloheximide, harvested by scraping, and lysed in 0.2 ml of lysis buffer containing 10 mM HEPES, 2.5 mM MgCl2, 62.5 mM KCl, 1% NP-40, 100 μg/ml cycloheximide, 500 μg/ml heparin, 0.2 U/ml RNasin, 1 mM DTT, and EDTA-free protease inhibitor (Roche). Lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4°C and subjected to sucrose density gradient centrifugation as described. After fractionation, proteins were precipitated using acetone and resuspended in SDS sample buffer.
COS7 cells were seeded 8–12 h before incubation in methionine- and cysteine-free DME medium supplemented with 5% fetal bovine serum (PAA Laboratories), 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all PAN Biotech) for 1 h. Then 200 μCi of 35S-labeled methionine and cysteine (EasyTag; PerkinElmer) was added to each dish, and cells were simultaneously exposed to cold shock at 10°C or kept at 37°C for different periods of time. As a control, one dish was treated with 500 μM Na arsenite for 1 h. Cells were then washed with PBS, collected, and solubilized in 150 μl of lysis buffer containing 15 mM Tris, pH 7.4, 15 mM MgCl2, 300 mM NaCl, and 1% Triton X-100. After centrifugation at 9000 rpm for 3 min, proteins were precipitated out of the supernatants by spotting 20 μl of each lysate onto Whatman paper and soaking in 5% trichloroacetic acid followed by acetone. The 35S incorporation was measured in 4 ml of Econofluor-2 (PerkinElmer) using a scintillation counter (LS 6000IC; Beckman Coulter, Brea, CA). For normalization, the total protein concentration of each sample was determined using the bicinchoninic acid protein assay reagent kit (Sigma-Aldrich). For each sample, 100 ng of total protein was resolved on a 5–20% polyacrylamide gradient Tris-glycine gel and stained using colloidal Coomassie. The gel was dried at 80°C for 2 h under vacuum, and 35S incorporation into newly synthesized proteins was detected by autoradiography using a phosphoimager (FLA-7000; FujiFilm, Tokyo, Japan).
Cap pull-down assay
Huh7 cells were seeded in 15-cm dishes and allowed to adhere for 8–12 h. To account for the reduced cell mass after cold shock, two dishes were subjected to 10°C cold shock for 10 h, one was treated with 0.2 μM rapamycin for 1 h, and one dish was kept under control conditions. Cells were lysed in 0.6 ml of cap binding buffer containing 50 mM Tris-HCl (pH 7.0), 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, and complete protease inhibitors (Roche). Lysates were cleared via centrifugation at 25,000 × g for 15 min at 4°C. A 50-μl amount of 7-methyl-GTP–conjugated Sepharose beads (GE Healthcare, Piscataway, NJ) was added to each sample. After rotation at 4°C for 2 h, beads were washed four times in a buffer containing 15 mM Tris-HCl (pH 7.2), 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40. Proteins were eluted in SDS sample buffer and separated on a 5–20% polyacrylamide gradient gel.
Measurement of ATP levels
After cold shock, cells were collected by trypsinization, and cell pellets were weighed on a fine balance before lysis in 20 mM Tris (pH 7.4), 0.1 mM EDTA, 2.67 mM MgSO4, and 33.3 mM DTT. Lysates were incubated on ice for 5–10 min and centrifuged at 13,000 rpm for 1 min. For ATP measurements, 20 μl of serially diluted lysates was added to 80 μl of lysis buffer containing 1 nM of firefly luciferase recombinantly expressed in E. coli and 500 μM luciferin. Light emission was measured with a luminometer (Lumat LB 9507; Berthold Technologies, Bad Wildbad, Germany). Results were normalized to the weight of the cell pellets and calculated as percentage of control.
Cell viability assay
Cells were subjected to cold shock for different time periods and treated with 20 μM compound C (Calbiochem, La Jolla, CA) or 20 μM Src inhibitor-1 (S2075; Sigma-Aldrich) where indicated. Cells were collected from the media and combined with those recovered from the dish by trypsinization, resuspended in 10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 1.8 mM CaCl2 and stained for 10 min on ice with 50 μg/ml propidium iodide (AppliChem, Darmstadt, Germany). Propidium iodide uptake was measured by flow cytometry using a FACSCanto II (BD Biosciences).