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Mammalian IRE1α dynamically and functionally coalesces with stress granules

Nature Cell Biology (2024)Cite this article

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Abstract

Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α–SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α–XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α–SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.

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Fig. 1: IRE1α clusters co-localize with SGs in response to different types of stress.
Fig. 2: Augmented association of IRE1α protein with SG components upon stress.
Fig. 3: Dynamic features of IRE1α–SG clusters at the ER.
Fig. 4: IRE1α’s cytosolic linker is essential for its association with SGs.
Fig. 5: IRE1α harbours a cytosolic linker with IDRs for clustering.
Fig. 6: Disruption of SG formation abolishes IRE1α clustering and blunts its functional activity.
Fig. 7: Enrichment of XBP1 mRNA splicing components in IRE1α–SG condensates.
Fig. 8: Mammalian IRE1α coalesces with SGs to form a workstation for more efficient ER stress handling.

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Data availability

For the IDR prediction analysis, protein sequences were obtained from the NCBI protein database with the following accession numbers: NP_001424.3 for hIRE1α, NP_031374.2 for hATF6α and NP_004827.4 for hPERK. For the sequence alignment analysis of IRE1 proteins from multiple species, protein sequences were from the Uniprot database with their Uniprot ID numbers as follows: O75460 for human, A0A2K5X0L5 for macaque, A0A0G2K2H4 for rat, Q9EQY0 for mouse, Q09499 for worm (Caenorhabditis elegans), A8JR46 for fruit fly (Drosophila melanogaster) and P32361 for yeast (Saccharomyces cerevisiae). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank P. Li from Tsinghua University for insightful discussions. This work was supported by grants from the Ministry of Science and Technology of China (National Key R&D Program of China 2018YFA0800700) and from the National Natural Science Foundation of China (82230026, 32021003, 92357307, 91857204 and 323B1006) to Y.L. and R.X. This work was also supported by the Fundamental Research Funds for the Central Universities (2042022dx0003) to Y.L.

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  1. These authors contributed equally: Songzi Liu, Xiaoge Zhang, Xin Yao.

Authors and Affiliations

  1. Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences; TaiKang Center for Life and Medical Sciences; the Institute for Advanced Studies; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China

    Songzi Liu, Xiaoge Zhang, Xin Yao, Shijia Huang, Peng Chen, Mingliang Tang, Jie Cai, Zhuyin Wu, Yiliang Zhang, Rongzhi Xu, Kai Liu, Yan Wang & Yong Liu

  2. Comprehensive Cancer Center, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA

    Guan Wang, Lei Jiang & Qiong A. Wang

  3. Department of Molecular & Cellular Endocrinology, Diabetes & Metabolism Research Institute, City of Hope Medical Center, Duarte, CA, USA

    Guan Wang, Lei Jiang & Qiong A. Wang

  4. Clinical Research Center, the Affiliated Hospital of Guizhou Medical University, Guiyang, China

    Jie Cai

  5. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Kangmin He

  6. University of Chinese Academy of Sciences, Beijing, China

    Kangmin He

  7. Department of Molecular and Integrative Physiology, the University of Michigan Medical School, Ann Arbor, MI, USA

    Liangyou Rui

  8. Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, China

    Jianmiao Liu

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Contributions

S.L., X.Z. and Y.L. conceived and designed the studies. S.L., X.Z. and X.Y. planned and performed most of the experiments and analysed the data. G.W., S.H., P.C., J.C., Z.W. and Y.Z. performed some of the cell line experiments. M.T., K.H. and R.X. assisted with microscopy imaging. K.L., Y.W., L.J., Q.A.W., L.R. and J.L. provided essential reagents and assisted with data analysis. S.L. and Y.L. wrote and edited the original manuscript.

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Correspondence to Yong Liu.

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Extended data

Extended Data Fig. 1 Endogenous IRE1α forms large clusters in response to different types of stress.

a. Representative confocal microscopy images of endogenous IRE1α clusters visualized by immunofluorescent (IF) staining of IRE1α (red) in HeLa cells treated with thapsigargin (Tg) at the indicated concentrations for 3 h. Nucleus was stained with DAPI (blue). Scale bar, 20 μm. Magnification of the demarcated region was shown as the inset for each condition from the merged images. Inset scale bar, 20 μm. Shown also is the quantification of the percentage of cells containing IRE1α clusters and the IRE1α cluster index (mean number in IRE1α cluster-positive cells). Results are presented as mean ± SD, n = 9 image fields analyzed for each condition from three independent experiments. b. Representative IF images of endogenous IRE1α clusters in HeLa cells treated with DMSO (Ctrl), Tg (1 μM, 3 h), sodium arsenite (SA, 500 μM, 1 h) or heat shock (HS, 43 °C, 1 h). Scale bar, 20 μm; Inset scale bar, 20 μm. The percentage of cells containing IRE1α clusters and the IRE1α cluster index are shown as mean ± SD, n = 15 image fields analyzed for each condition from three independent experiments.

Source data

Extended Data Fig. 2 IRE1α but not PERK or ATF6 forms SG-colocalized clusters in response to different types of stress.

a-c. HeLa cells were treated with DMSO control (Ctrl), 1 μM thapsigargin (Tg, 3 h), 500 μM sodium arsenite (SA, 1 h) or heat shock (HS, 1 h) at 43 °C. a. Representative confocal microscopy images of IRE1α clusters and stress granules (SGs) visualized by IF staining of IRE1α (red) along with the SG marker TIA1 (green), with enlargement of the indicated inset areas shown from the merged images. Scale bar, 10 μm. The arrow in each enlarged inset indicates the plane for generating line profiles, and the line profiling of fluorescence intensities is shown on the right with Pearson’s correlation coefficient (r) included. Results are representative of three independent experiments for each condition. b. Colocalization of immunofluorescent signal for IRE1α with that for TIA1. Pearson’s correlation coefficient values above the threshold are shown as mean ± SD, n=number of line profiles analyzed from three independent experiments. c. Quantification of the percentage of cells containing colocalized IRE1α-SG clusters and the IRE1α-SG index (the mean colocalized cluster number in IRE1α-SG-positive cells). Data are shown as mean ± SD, n = 16 image fields analyzed for each condition from three independent experiments. d, e. Mouse embryonic fibroblast (MEF) cells (d) or HEK293T cells (e) were treated with DMSO control (Ctrl), 1 μM Tg for 3 h, 500 μM SA for 1 h or HS for 1 h at 43 °C. Representative confocal images of IRE1α clusters (red) and SGs by IF staining of G3BP1 (green). Shown also are magnifications of the indicated areas from the merged images. Scale bar, 10 μm (d, e). f-h. PERK or ATF6α does not form SG-associated clusters. f. Diagram showing the prediction of disordered regions of human PERK and ATF6α protein by PONDR (Predictor of Natural Disordered Regions, VL-XT). g, h. Representative confocal IF images of G3BP1 (green) and C-Myc-tagged PERK (red, g) in transfected HeLa cells, or G3BP1 (green) and endogenous ATF6α (red, h) in HeLa cells, following exposure to the indicated stressors. Shown also on the right are fluorescent intensity profiles of the indicated planes, and results are representative of three independent experiments for each condition. Scale bar, 10 μm.

Source data

Extended Data Fig. 3 Analysis of the formation of IRE1α-SG clusters in response to hypoxia and glucose deprivation.

a, b. Representative confocal images by IF staining of IRE1α (red) along with G3BP1 (green) in HeLa cells upon exposure to (a) hypoxia (1% O2) or (b) glucose deprivation (GD) for the indicated time intervals. Scale bar, 20 μm. Inset scale bar, 10 μm. c. Representative confocal IF images of IRE1α (red) along with G3BP1 (green) in HeLa cells cultured in glucose-free medium (GD) without (Ctrl) or with 25 mM 2-deoxy-D-glucose (2-DG) for 1 h. Scale bar, 20 μm. Inset scale bar, 10 μm. Shown also is the quantification of the percentage of cells with IRE1α, SG or colocalized IRE1α-SG clusters. Results are presented as mean ± SD, and n=number of image fields analyzed from three independent experiments.

Source data

Extended Data Fig. 4 Coordinated reductions of coalesced IRE1α-SG clusters during stress recovery.

a-c. HeLa cells were treated with (a) 1 μM thapsigargin (Tg, 3 h), (b) 500 μM sodium arsenite (SA, 1 h), or (c) heat shock (HS, 1 h) at 43 °C. Cells were then allowed to recover from stress by culturing in the medium without stressors for the indicated time intervals. Representative confocal images of IRE1α clusters (red) and G3BP1-positive SGs (green) by IF staining. Shown also are merged images with DAPI staining. Scale bar, 20 μm. Shown below the images is quantification of the percentage of cells with IRE1α, SG or colocalized IRE1α-SG clusters at the indicated post-stress time. Results are presented as mean ± SD, and n = 9 image fields analyzed for each condition from three independent experiments.

Source data

Extended Data Fig. 5 The ER lumenal domain of IRE1α is also crucial for its clustering with SGs.

a. Representative time-lapse imaging of light-activated clustering of IRE1α-mCherry-CRY2 without (FL) or with deletion of its lumenal domain (ΔLD) in live COS7 cells. Scale bar, 10 μm. Magnifications of images are also shown for the indicated inset regions. Inset scale bar, 5 μm. b. Representative images of live tagBFP-KDEL-expressing COS7 cells transfected with the indicated ddFP constructs following treatment with 1 μM Tg for 30 min. Scale bar, 10 μm. Quantification of the number of ddFP condensates per cell is also shown as mean ± SD, and n=number of cells from three independent experiments. ***P < 0.001 by two-sided unpaired t-test. c. Representative images of IRE1α (green) colocalization with G3BP1 (red) in HeLa cells transfected with the IRE1α-YFP construct or its ER lumenal domain deletion (ΔLD) variant following treatment with 1 μM Tg for 3 h. Scale bar, 20 μm. Inset scale bar, 10 μm. Shown on the right are line profiles of fluorescence intensities of the indicated plane by the white arrow for each merged image, and results are representative of three independent experiments for each condition.

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Extended Data Fig. 6 Depletion of IRE1α does not affect SG formation or its tethering connection to the ER.

a. Representative confocal microscopy IF images of TIA1- (green) and G3BP1-positive (red) granules (left panels) as well as G3BP1 (green) and IRE1α (red) (right panels) in WT HeLa cells versus a HeLa cell line with CRISPR-Cas9-based knockout of IRE1α (IRE1α-KO) following treatment with DMSO (Ctrl), Tg (1 μM, 3 h), SA (500 μM, 1 h) or HS (43 °C, 1 h). Shown also are magnifications of the indicated inset areas from the merged images. Scale bar, 20 μm. b. WT versus IRE1α-KO HeLa cells were transfected with tagBFP-KDEL along with CalN-RA and GB-G3BP1 ddFP constructs to monitor the contacts between the ER and SGs. Representative live cell images acquired following DMSO or Tg (1 μM, 30 min) treatment. Scale bar, 20 μm. Inset scale bar, 1 μm. Quantification of Tg-induced ddFP signals per cell is shown as mean ± SD on the right, and n=number of cells from 2 independent experiments. n.s., no significant difference by two-sided unpaired t-test.

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Extended Data Fig. 7 Blocking SG formation by cycloheximide abolishes IRE1α enrichment in the RNP granule fractions.

HEK293T cells were treated with DMSO control (-), (a) Tg (1 μM, 3 h), (b) SA (500 μM, 1 h) or (c) HS (43 °C, 1 h) following no cycloheximide (CHX) pretreatment or 10 μg/ml CHX pre-treatment. Immunoblot analysis of the indicated proteins from whole cell lysate (Input), the soluble fraction without nucleus (Soluble), and the insoluble RNP granule (RG) fraction. α-Tubulin was used as the loading control. Relative abundance of the indicated proteins in the RG fraction was quantified and normalized to their input level. Results are shown as mean ± SD, and n = 3 independent experiments for each condition. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test.

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Extended Data Fig. 8 Effects of small-molecule protein translation, IRE1α Kinase/RNase or ISR inhibitors upon IRE1α-SG clusters.

a-c. Effects of protein translation or IRE1α Kinase/RNase inhibitors. HeLa cells were pre-treated for 1 h with DMSO, cycloheximide (CHX, 10 μg/ml), emetine (Eme, 10 μg/mL), puromycin (Puro, 10 μg/ml), the IRE1α RNase inhibitor 4μ8C (10 μM) or IRE1α kinase inhibitor KIRA6 (5 μM) before 3 h Tg treatment. a. Representative confocal IF images of IRE1α clusters (red) and G3BP1-positive SGs (green). Scale bar, 10 μm. Shown also is the quantification of the percentage of cells containing colocalized IRE1α-SG clusters and the IRE1α-SG cluster index. b. Representative agarose gel showing RT-PCR analysis of XBP1 mRNA splicing levels. c. Representative immunoblot analysis of IRE1α and the indicated SG marker proteins. d. Effects of ISRIB on the formation of IRE1α-SG clusters. Representative confocal IF images of IRE1α (red) and G3BP1 (green) in HeLa cells that were pre-treated with DMSO control or ISRIB (1 μM) and then treated with Tg (1 μM, 3 h), SA (0.2 mM, 1 h) or HS (43 °C, 1 h). Scale bar, 20 μm. Inset scale bar, 10 μm. e. Quantification of the percentage of cells containing IRE1α clusters, SGs or colocalized IRE1α-SG clusters in (d). Data in a, e are shown as mean ± SD, and n=number of image fields analyzed from three independent experiments for each condition. ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test.

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Extended Data Fig. 9 Disruption of SG formation abolishes IRE1α clustering and blunts its functional activity.

a-c. Formation of IRE1α clusters was abrogated in G3BP1/2-deficient cells. a. Representative IF images of IRE1α clusters (red) and TIA1-positive SGs (green) in WT or G3BP1/2-dKO HeLa cells following treatment with DMSO (Ctrl), Tg (1 μM, 3 h), SA (500 μM, 1 h) or HS (43 °C, 1 h). Scale bar, 10 μm. b. Representative IF images of IRE1α clusters (red) and G3BP1-positive SGs (green) in WT or G3BP1/2-dKO #1 and #2 HeLa cells following treatment with DMSO (Ctrl), SA (500 μM, 1 h) or HS (43 °C, 1 h). Scale bar, 10 μm. c. Bar graphs showing quantified percentages of cells containing IRE1α clusters from (b). Data are presented as mean ± SD, n=number of image fields analyzed from three independent experiments for each condition. *** indicates all tested P values are <0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. d-g, Ablation of G3BP1/2 resulted in lower activation of the IRE1α-XBP1 pathway. d, e. Immunoblot analysis of XBP1s and the indicated proteins from lysates of WT or G3BP1/2-dKO HeLa cells following treatment with (d) Tg (1 μM, 3 h) or (e) HS (43 °C, 1 h). The abundance of XBP1s protein was quantified by normalizing to α-Tubulin. f. Quantitative RT-PCR analysis of the mRNA abundance of the indicated XBP1s target genes. Results in d, e and f are presented as mean ± SD, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. g. Immunoblot analysis of the indicated proteins in WT or G3BP1/2-dKO HeLa cells transfected with the empty vector (Vec) or XBP1s construct prior to treatment with the indicated stressors.

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Extended Data Fig. 10 Formation of SGs is required for IRE1α clustering and its enhanced RNase output.

a-c. Restored expression of G3BP1/2 normalized the formation of IRE1α-SG clusters in G3BP1/2-deficient cells. a. Representative IF images of IRE1α (red), G3BP1 (green) and G3BP2 (blue) in G3BP1/2-dKO HeLa cells that were transiently transfected with both G3BP1 and G3BP2 constructs prior to treatment with DMSO (Ctrl) or Tg (1 μM, 3 h). Scale bar, 10 μm. b, c. WT and G3BP1/2-dKO HeLa cells were transiently transfected with empty vector (Vec) or the G3BP1/2 constructs for 24 h, followed by treatment with Tg (1 μM, 3 h in b; 10 μM in c). b. Immunoblot analysis of the indicated proteins. c. Trypan blue staining analysis. Quantification of the percentage of dead cells shown as mean ± SD, n = 3 independent experiments for each condition. **P < 0.01, ***P < 0.001, n.s., no significant difference by two-way ANOVA with Tukey’s multiple comparisons test. d, e. HeLa cells were transiently transfected with scramble control RNA or siRNAs against UBAP2/2 L for 48-72 h, followed by treatment with the indicated stressors. d. Quantitative RT-PCR analysis of the mRNA abundance of the indicated genes. Results are presented as mean ± SD, n = 3 independent experiments. ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. e. Representative confocal IF images of IRE1α (red), UBAP2/2 L (green) and G3BP1 (blue) in HeLa cells. Scale bar, 20 μm. Inset scale bar, 10 μm. f-h. HeLa cells were pre-treated with DMSO or Mitoxantrone (10 μM, 1 h) followed by Tg treatment (1 μM, 3 h). f. Representative confocal IF images of IRE1α (red) and G3BP1 (green). Scale bar, 20 μm. Inset scale bar, 10 μm. g. Quantitative RT-PCR analysis of XBP1 mRNA splicing. Data are presented as mean ± SD, n = 3 independent experiments. ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. h. Immunoblot analysis of the indicated proteins.

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Supplementary information

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Supplementary Video 1

Live time-lapse image of an IRE1α–SG cluster undergoing fission at the ER.

Supplementary Video 2

Live time-lapse image of an IRE1α–SG cluster undergoing fusion at the ER.

Supplementary Video 3

Live time-lapse image of light-activated clustering of IRE1α-FL-mCherry-CRY2 versus IRE1α-ΔL-mCherry-CRY2 in the absence of stress.

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Liu, S., Zhang, X., Yao, X. et al. Mammalian IRE1α dynamically and functionally coalesces with stress granules. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01418-7

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