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First published online February 4, 2009
doi: 10.1242/10.1242/jcs.038208

Journal of Cell Science 122, 563-573 (2009)
Published by The Company of Biologists 2009
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Mammalian Staufen 1 is recruited to stress granules and impairs their assembly

María Gabriela Thomas1,2,3,*, Leandro J. Martinez Tosar1,3,*, María Andrea Desbats1,3,*, Claudia C. Leishman1 and Graciela L. Boccaccio1,2,3,{ddagger}

1 Fundación Instituto Leloir, Av. Patricias Argentinas 435, Buenos Aires, Argentina
2 IIBBA-CONICET, Av. Patricias Argentinas 435, Buenos Aires, Argentina
3 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina


Figure 1
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  		Fig. 1. Stau1 is recruited to stress granules upon induction of ER or oxidative stress. NIH 3T3 (A) and HeLa cells (B) were exposed to 1 µM thapsigargin or 0.5 mM sodium arsenite for 1 hour and immunostained for Stau1 and the indicated stress granule markers. (C) BHK cells were exposed to arsenite in the presence of cycloheximide (CHM) or puromycin (Pur). Representative cells showing inhibition of stress granule formation by cycloheximide and the absence of effect by puromycin are depicted. In the absence of stress granules, Stau1 remained dispersed in the cytoplasm. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. Stau1 is not recruited to processing bodies. (A,B) NIH 3T3 cells were continuously exposed to thapsigargin and stained for TIAR and DCP1a at the indicated times. Percentage of cells with stress granules is indicated. Scale bar: 10 µm. The intensity profiles of Dcp1a and TIAR were analyzed by confocal line-scanning of single processing bodies and stress granules (B). Processing bodies were initially detected in close apposition with stress granules (1 hour). At 4 hours, larger stress granules were observed and Dcp1a staining was detected both in processing bodies and as punctuated inclusions inside stress granules. At 8 hours, free processing bodies were absent and Dcp1a signal was detected in the remaining stress granules. (C) Representative processing bodies showing a lack of signal (71-77% of processing bodies) or weak signal (23-29% of processing bodies) of endogenous or transfected Stau1 under resting conditions. Bottom, immunostaining with specific antibodies showed that PABP is excluded from processing bodies upon oxidative stress induction. Scale bars: 1 µm. (D) U2OS (a) or NIH 3T3 cells (b) were transiently transfected with the indicated constructs for 24 hours. After treatment with arsenite for 1 hour, cells were immunostained for TIA-1 and Dcp1a, Stau1 or Xrn1. The presence of Stau1 or Xrn1 in randomly selected processing bodies (PBs) from 10 cells in each condition was evaluated using x100 confocal images and Z-slice analysis. The percentage of processing bodies containing the indicated markers is plotted.

 

Figure 3
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  		Fig. 3. Stau1 depletion facilitates stress granule formation. (A) Stau1-ECFP or Stau2-ECFP constructs were independently transfected into COS-7 cells simultaneously with the indicated siRNAs (NR, non-relevant). Expressing cells and total cells identified by DAPI staining were counted 16 hours after transfection. (B) NIH 3T3 cells were treated with siNR or siStau1 and extracts were analyzed by western blot. Intensity of the Stau1 signal relative to that of β-actin indicates a 75% reduction of Stau1 levels. (C) Line-scan analysis of arsenite-induced stress granules in siNR- or siStau1-treated cells indicates the presence of negligible amounts of Stau1 upon Stau1 depletion. Scale bar: 1 µm. (D) Upon treatment with the indicated siRNAs, cells were continuously exposed to arsenite and cells containing stress granules (SGs) were identified by TIAR staining. A representative experiment out of three is shown where approximately 200 siNR-treated cells and 300 siStau1-treated cells randomly selected from duplicate coverslips were analyzed for each time point. Stress granule formation is facilitated in Stau1-depleted cells; ***P<0.0001 for each data pair. (E) TIAR and Stau1 staining in siNR- and siStau1-treated cells after 1 hour arsenite treatment. Percentage of cells containing stress granules is indicated. (F,G) After treatment with the indicated siRNA, NIH 3T3 cells were exposed to thapsigargin and stained for TIAR and Stau1. (F) stress granule size was evaluated in approximately 1800 stress granules present in 90 cells at 40 or 60 minutes after thapsigargin treatment. (G) Time course of stress granule formation. A representative experiment out of three is depicted. A minimum of 400 cells was analyzed for each point. Stau1 depletion significantly facilitates the formation of stress granules induced by ER stress (***P<0.0003). (H) NIH 3T3 cells were treated with the indicated siRNAs and continuously exposed to 100 nM thapsigargin. Percentage of viable cells was determined in triplicate using the MTT viability assay. A representative experiment out of three is depicted. ***P<0.0001.

 

Figure 4
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  		Fig. 4. Stau1 overexpression inhibits stress granule formation. (A,B) COS-7 cells were transfected with Stau1-V5 and exposed to arsenite 24 hours after transfection. Representative cells showing the presence of stress granules in non-expressing or low expressing cells and their absence upon moderate Stau1-V5 overexpression are shown. Percentage of transfected or non-transfected cells containing stress granules is indicated. Scale bars: 10 µm. (C) NIH 3T3 cells were continuously exposed to thapsigargin 16 hours after transfection with Stau1-ECFP or ECFP. Cells with moderate level of expression (less than four times the endogenous levels) were analyzed for stress granule formation. A minimum of 120 transfected cells or neighboring non-transfected cells (NTECFP and NTStau1) from duplicate coverslips were analyzed. ***P<0.0001 for Stau1-ECFP expressing cells. (D) U2OS (a-c); NIH 3T3 (d); H1299 (e) Cos7 (f) or HeLa cells (g) were transfected with the indicated Stau1 construct and exposed to 250 nM or 500 nM thapsigargin (a and b); or to 0.1, 0.2 or 0.25 mM arsenite (c, d and e, respectively); or to 0.5 mM arsenite (f and g). Approximately 200 transfected and non-transfected cells were counted in each case. Stress granule formation is expressed as the ratio of the percentage of transfected stress granule-forming cells relative to that of neighboring non-transfected cells. The incidence of stress granules in non-transfected cells was as follows: a, 32%; b, 54%; c, 41%, d, 86%; e, 35%; f, 97% and g, 86%. Differences between averaged independent experiments (two in b and f) or between replicate coverslips (a-g) were less than 10% in all cases. (E) U2OS cells transfected with the indicated constructs were exposed to thapsigargin (upper panel) or arsenite (bottom panel). Representative stress granules showing inclusion of RBD234-EGFP or exclusion of TBD-RBD5-EGFP are shown. Scale bars: 10 µm (left panels) and 2 µm (insets).

 

Figure 5
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  		Fig. 5. Stau1 modulates stress granule assembly downstream of eIF2{alpha} phosphorylation. (A) U20S cells were transfected with Stau1-ECFP and exposed to 1 µM hippuristanol for 60 minutes. (B) Stress granule formation was evaluated in 350 Stau1-ECFP- or ECFP-transfected cells and in 300 neighboring non-transfected cells (NT) treated as in A. Stau1-ECFP inhibits stress granule formation (***P<0.0001). (C) Cells were continuously exposed to arsenite, stress granules were visualized by TIAR staining and the levels of phosphorylated eIF2{alpha} relative to β-actin were determined by western blot of 2.5, 5 or 10 µg total protein. Total levels of eIF2{alpha} remained constant (not shown). Phosphorylation of eIF2{alpha} peaked simultaneously with stress granule formation and thereafter decayed below basal levels. (D) Levels of phosphorylated or total eIF2{alpha} were determined by immunofluorescence in single cells expressing Stau1-ECFP under control conditions (C; n=26) or upon arsenite exposure (Ars; n=51) and in neighboring non-expressing cells (NT) in control (n=32), or stress conditions (n=64).

 

Figure 6
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  		Fig. 6. Stau1 associates with stress-resistant polysomes. (A) NIH 3T3 cells were exposed to 0.5 mM arsenite for 1 hour, polysomes were separated in sedimentation gradients and fractions were analyzed by western blot to detect P0, a marker for large ribosomal subunits, S6, a marker for small subunits, PABP, TIAR and Stau1. (B) Distribution of P0 and Stau1 in pooled fractions from the gradient shown in A were evaluated by western blot and relative abundances are represented in the graphs. (C) Three independent experiments were performed as in A, and the distribution of polysomes was evaluated by following the P0 and S6 distributions in the gradient. Left column pair, total number of fractions = 13; polysome-containing fractions = 10-13. Middle and right column pairs, total number of fractions = 24; polysome-containing fractions = 16-24. The content of Stau1 in the polysomal fraction was measured by western blot analysis and is expressed normalized to S6 (left column pair) or P0 (middle and right column pairs). Duplicate western blot analysis of each gradient showed variations less than 10%. On average, the polysomes that remain upon stress induction contain twice the amount of Stau1 than found in polysomes under resting conditions. (D,E) Cells were transfected with ECFP or Stau1-V5 and exposed to 0.5 mM arsenite for 1 hour. The polysome profile was evaluated by monitoring the distribution of P0 in the gradient and the relative amount of polysomes is expressed as the percentage of P0 in the polysomal fraction relative to total. The amount of polysomes recovered after stress induction was increased from 10 to 20% in the presence of Staufen1-V5. (F) A model for the modulation of stress granules by Stau1. Under resting conditions, Stau1 is associated with polysomes by binding to mRNAs and ribosomal subunits. Cellular stress or pharmacological inhibition of 60S ribosomal recruitment provokes the breakdown of polysomes and concomitant accumulation of abortive translation initiation complexes that are aggregated by specific RBPs including TIA-1, TIAR and G3BP and by the O-glycosylation of ribosomal proteins (reviewed by Anderson and Kedersha, 2008Go; Ohn et al., 2008Go). Stau1 is recruited to growing stress granules by a piggyback mechanism. Formation of stress granules is counterbalanced by Stau1, which stabilizes polysomes against stress-induced breakdown, thus helping stress granule dissolution. In addition, Hsp70 contributes to stress granule disassembly (Mazroui et al., 2007Go).

 

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© The Company of Biologists Ltd 2009