FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress

Abstract

FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS-associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non-disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re-distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress.

Figure 1. Endogenous FUS redistributes to the cytoplasm and localizes to cytoplasmic stress granules in response to sorbitol

Confocal images of untreated HeLa cells (top row in each panel) as compared to cells treated with 0.4 M sorbitol for 1 hr (bottom row in each panel; A-C) are shown. Cells probed with an anti-FUS antibody (green) and either the stress granule marker anti-G3BP (A) or anti-TIAR (B) revealed that FUS co-localizes with stress granules in response to sorbitol (see also supplementary material Movies M1 and M2). (C) P-bodies were detected by anti-GE-1/hedls antibody in both untreated and treated conditions; however, the majority of P-bodies did not exhibit co-localization with FUS (see also Supplementary Material Movie M3). Cells were counter stained with the nuclear marker DAPI (blue; A-C). Images are representative of at least n=3 experiments. Scale bar represents 10 μm.

Figure 2. The recruitment of FUS to cytoplasmic stress granules is stress-specific

HeLa cells were treated with either 0.5 mM sodium arsenite (NaAsO₂) for 1 hr, 1.5 mM hydrogen peroxide (H₂O₂) for 2 hrs, 50 μM thapsigargin for 30 min, or heat shock at 43 °C for 30 min. Immunofluorescence revealed that G3BP-positive stress granules (red) formed under all stress conditions. FUS (green) remained nuclear and absent from stress granules under these stress conditions, similar to the unstressed condition (top panel). Nuclei were stained with DAPI (blue). All images are representative of n=3 independent experiments. Scale bar represents 10 μm.

Figure 3. The response of FUS to sorbitol is rapid and reversible

(A) A representative time-course for the cytoplasmic redistribution of FUS into stress granules upon exposure to hyperosmolar stress. HeLa cells were treated with 0.4 M sorbitol for the indicated time points, fixed, and assessed by immunofluorescence with anti-FUS (green) and anti-G3BP (red) antibodies, and the nuclear marker DAPI (blue). Elevated levels of cytoplasmic FUS were detected as early as 10 min. FUS accumulated into discreet stress granules by 20 min. The nucleo-cytoplasmic distribution of FUS continued to shift towards the cytoplasm over the remaining time course. (B) A representative time-course for the return of FUS to the nucleus and the concomitant disassembly of stress granules upon withdrawal of sorbitol. HeLa cells were treated with 0.4 M sorbitol for 1 hr, after which the sorbitol was replaced with fresh media and the cells were processed as described in (A). A majority of FUS re-localized to the nucleus within 10 min. Some G3BP positive stress granules persisted for up to 1 hr. Images are representative of at least n=3 experiments. Scale bar represents 10 μm.

Figure 4. An inhibitor of stress granule assembly prevents the cytoplasmic redistribution of FUS, though stress granules still assemble in the absence of FUS

(A) HeLa cells were treated with 0.4 M sorbitol for 1 hr, 50 μg/ml emetine for 1 hr or pre-treated with emetine followed by sorbitol treatment. Cells were then fixed and probed by immunofluorescence for DAPI (blue), FUS (green) and G3BP (red). Emetine pre-treatment inhibited both stress granule assembly, as evidenced by the diffuse G3BP signal, and the cytoplasmic redistribution of FUS in the presence of sorbitol. (B) HeLa cells were transfected with non-targeting siRNA (siNT) or siRNA against FUS (siFUS) for 48 hrs, subsequently treated with 0.4 M sorbitol for 1 hr, and then processed for immunofluorescence as described above. Cells treated with either siFUS or siNT exhibited normal stress granule formation (B, red) in response to sorbitol, despite a significant reduction in FUS protein levels in siFUS treated cells as evidenced by immunofluorescence (green; B) and western blot (C). All images are representative of at least n=3 independent experiments. Scale bar represents 10 μm.

Figure 5. Methylation regulates the nucleo-cytoplasmic distribution of FUS

(A,B) HeLa cells were treated with 0.4 M sorbitol for 1 hr, or pre-treated with 50 μM AdOx for 24 hrs prior to sorbitol treatment (AdOx + sorbitol) and subjected to confocal immunofluorescence imaging with anti-FUS (green) and anti-G3BP (red) antibodies. Sorbitol decreased the percentage of cellular FUS in the nucleus from 90±5.1% in untreated cells to 46.5±9.8%. Pre-treatment of cells with AdOx prior to sorbitol increased the percentage of cellular FUS in the nucleus to 75.6±7.4%. Data shown are the average of three independent experiments ± standard deviation. Statistical significance was determined by ANOVA and Tukey's post-hoc pairwise test (**p<0.005, ***p<0.0005). No other comparisons were statistically significant. Scale bar represents 10 μm. (C) FUS was immunoprecipitated from untreated HeLa cells or from AdOx + sorbitol cells and probed with the ASYM24 antibody by western blot. FUS was used as a loading control. (D) Densitometry analysis of (C) revealed a 68.6±7.8% decrease in the amount of FUS that is arginine dimethylated when cells were pre-treated with AdOx compared to untreated cells. Data shown are the average of three independent experiments ± standard deviation. Statistical significance was determined by Student's t-test (** p<0.005).

Figure 6. Methylation does not regulate the incorporation of FUS into stress granules

HeLa cells were transiently transfected to express GFP-FUS G515X. Cells were exposed to 0.4 M sorbitol for 1 hr either (A) in the absence of AdOx or (B) after cells had been pre-treated with 25 μM AdOx for 8 hrs. (A) Confocal imaging showed that GFP-FUS G515X (green) assembles into G3BP-positive stress granules (red) upon sorbitol treatment (top panel). Co-staining with the ASYM24 antibody (a far-red fluorescence probe was employed; green is used in the images for clarity) revealed that these same stress granules contained asymmetrically dimethylated proteins (bottom panel). (B) While the ASYM24 signal is dramatically decreased within stress granules and cells pre-treated with AdOx (bottom panel), there is still a robust association of GFP-FUS with stress granules under the same conditions (top panel). Scale bar represents 10 m. (C) Immunoprecipitation of GFP-FUS G515X with an anti-GFP antibody and a subsequent western blot analysis with ASYM24 revealed that FUS is hypomethylated due to AdOx pretreatment. All data are representative of n=3 independent experiments.

Figure 7. Reduced FUS expression causes cells to become susceptible to sorbitol induced toxicity

(A) Expression of either a non-targeting scrambled shRNA (shSC) or shRNA against FUS (shFUS) was induced by doxycycline for 48 hrs in NSC-34 cell lines, resulting in ~70% knock-down of the FUS protein as determined by western blot. Tubulin was used as a loading control. Cells were then treated with 0.4 M sorbitol or 0.25 mM sodium arsenite for 8 hrs and subjected to the (B) MTT cell viability assay or (C) LDH cell toxicity assay. (B) A significant decrease in cell viability was detected in shFUS cells (38±6%) compared to shSC cells (76±11%) when treated with sorbitol, whereas shFUS cells did not exhibit an analogous susceptibility to sodium arsenite (59±4% for shFUS versus 57±6% for shSC). (C) A higher percentage of cell death was detected in shFUS cells (15.2±3.6%) compared to shSC cells (4.2±1.4%) in response to sorbitol, whereas no difference in cell death was detected when these lines were stressed with sodium arsenite (7.4±2.2% for shFUS versus 4.3±0.7% for shSC). (B,C) Data shown are an average from n=3 independent experiments ± standard deviation. Statistical significance was determined by Student's t-test (** p<0.005).