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The STING phase-separator suppresses innate immune signalling

Nature Cell Biology volume 23, pages 330–340 (2021)Cite this article

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Abstract

Biomolecular condensates (biocondensates) formed via liquid–liquid phase-separation of soluble proteins have been studied extensively. However, neither the phase-separation of endoplasmic reticulum (ER) transmembrane protein nor a biocondensate with organized membranous structures has been reported. Here, we have discovered a spherical ER membranous biocondensate with puzzle-like structures caused by condensation of the ER-resident stimulator of interferon genes (STING) in DNA virus-infected or 2′3′-cGAMP (cyclic GMP-AMP)-treated cells, which required STING transmembrane domains, an intrinsically disordered region (IDR) and a dimerization domain. Intracellular 2′3′-cGAMP concentrations determined STING translocation or condensation. STING biocondensates constrained STING and TBK1 (TANK binding protein 1) to prevent innate immunity from overactivation, presumably acting like a ‘STING-TBK1-cGAMP sponge’. Cells expressing STING-E336G/E337G showed notably enhanced innate immune responses due to impaired STING condensation after viral infection at later stages. Microtubule inhibitors impeded the STING condensate gel-like transition and augmented type I-interferon production in DNA virus-infected cells. This membranous biocondensate was therefore named the STING phase-separator.

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Fig. 1: Viral infection or 2′3′-cGAMP induces STING ER-associated biocondensates.
Fig. 2: cGAMP induces STING condensation and a fluid-to-gel transition.
Fig. 3: cGAMP or Mn2+ promotes STING phase-separation and the gel-like transition in vitro.
Fig. 4: STING ER-associated condensates have puzzle-like structures.
Fig. 5: STING puzzle-like structures are formed via two routes.
Fig. 6: The STING puzzle-like structure insulates STING-TBK1 from IRF3.
Fig. 7: The STING condensate prevents overactivation of innate immunity.
Fig. 8: STING condensation was affected by STING-autoimmune mutations and its gel-like transition was inhibited by microtubule inhibitors.

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All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank members from the Core facility at the National Center for Protein Sciences at Peking University for assistance with electronic microscopy, optical microscopy, flow cytometry and LC-MS, in particular Y. Liu, Y. Hu, H. Zhang, Y. Jiang, P. Dong and Y. Xie for invaluable technical help with electronic microscopy, C. Shan, H. Lv, S. Qin and X. Li for invaluable technical help with optical microscopy, L. Du and H. Yang for invaluable technical help with flow cytometry and H. Li and D. Liu for invaluable technical help with LC-MS/MS. We thank X. Ji, M. Fang, J. Xi, J. Chen, R. Wang and R. Zhou for viruses, plasmid and assistance with the optoDroplets assay. This work was supported by the National Natural Science Foundation of China (31830022 and 81621001) and the Chinese Ministry of Science and Technology (2019YFA0508500 and 2020YFA0707800).

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Authors and Affiliations

  1. Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking University, Beijing, China

    Xiaoyu Yu, Liyuan Zhang, Yanfang Zhai, Qifei Jiang, Mengran Yi, Ziran Ruan, Run Fang, Zhaolong Chen, Xiaohan Ning & Zhengfan Jiang

  2. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Xiaoyu Yu, Liyuan Zhang, Yanfang Zhai, Qifei Jiang, Mengran Yi, Xiaobing Deng, Ziran Ruan, Run Fang, Xiaohan Ning & Zhengfan Jiang

  3. Center for Quantitative Biology, Peking University, Beijing, China

    Jingxiang Shen

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Contributions

X.Y. and Z.J. designed the research. X.Y. performed the experiments. L.Z., Y.Z., Q.J., M.Y., Z.R., X.D., Z.C., R.F. and X.N. assisted with the experiments. J.S. assisted with the cubic membrane research. X.Y. and Z.J. analysed the data and wrote the manuscript.

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Correspondence to Zhengfan Jiang.

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Extended data

Extended Data Fig. 1 Virus infection or 2’3’-cGAMP induces STING ER-associated condensate formation.

a, STING expression in HeLa TMEM173−⁄−-STING-GFP cell (hereon HeLa-STING-GFP), COS-7-STING-GFP, THP-1 cell and HT1080 cell was analyzed by western blot using antibodies against STING and GAPDH. b, HeLa-STING-GFP and COS-7-STING-GFP cells were stimulated with VACV or cGAMP as in Fig. 1a. The indicated proteins were analyzed at the indicated times by western blot with indicated antibodies. c, Representative images of STING condensate induced by cGAS activation. HeLa -STING-GFP were treated with 2 μg/ml 2’3’-cGAMP for 1 h or infected with indicated virus for 18 h. d, e, COS-7 cells expressing STING-GFP, mCherry-KDEL and LAMP1-iRFP (d) and COS-7 cells expressing STING-GFP, mCherry-KDEL and LC3B-iRFP (e) were activated by cGAS-transfection for 72 h. Representative confocal images showed STING condensates were co-localized with lysosomes (d) and autophagosomes (e). 293T cells transfected with plasmid expressing cGAS and STING-GFP for 72 h were stained by OTO method and analyzed by TEM. Representative images of puzzle-like structures digested by lysosomes (d) and autophagosome (e) were shown. f, 3D reconstruction of STING condensates in different cells. 293T and COS-7 cells were transfected with plasmid expressing cGAS and STING-GFP for 18 h. HeLa TMEM173−⁄−-STING-GFP and COS-7-STING-GFP cells were treated with VACV or 2’3’-cGAMP for 18 h or 1 h respectively. Series z-stack images of live cells were captured by confocal microscope and then 3D reconstruction was performed. Typical optical sections of z-stack were shown. g, COS-7 and 293T cells were transfected with plasmid expressing cGAS, STING-GFP and mCherry-KDEL. Live cell confocal images were taken. For all Extended Data Fig. 1, data were representatives of at least three independent experiments. Confocal images were representatives of fields, in which >95% of the cells displayed similar pattern. TEM images represented at least five cells with the same pattern. Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images in source data.

Source data

Extended Data Fig. 2 2’3’-cGAMP induces STING condensation and fluid-to-gel transition.

a, Representative confocal images of STING condensate fusion (white box). HeLa-STING-GFP were activated by 2’3’-cGAMP for 1 h (top) or 293T cells were transfected with plasmid expressing STING-GFP for 18 h (bottom). b, COS-7 cells were transfected with plasmid expressing STING-GFP. FRAP analysis of STING condensates was performed. ER membrane structures of a representative condensate were shown and zoomed (red box). ER membrane structures changed dramatically within every 6 seconds, as none of two zoomed images had same membrane distribution. c, STING condensates grew larger with increased STING protein expression. 293T cells were transfected with plasmid expressing STINGWT-GFP or STINGd309-379-GFP respectively. After 12 h, cells with similar fluorescence intensity were chosen for live cell movie. Representative images of same cell at indicated times were shown. d, 293T cells were transfected with plasmid expressing STING-mNeonGreen (top) or STING fused with GFP at N-terminus (bottom) for 18 h. Representative confocal images of STING-mNeonGreen condensate appearance (yellow arrows), fission and fusion (red arrows) were shown. Representative FRAP images of GFP-STING were taken before and after photobleaching at the indicated times. Zoomed image of representative STING condensates showed organized puzzle-like structure. Quantification of FRAP on STING condensates over the indicated time course was shown (right, mean ± SD, n = 5 STING condensates). e, Blue-light activation of 293T cells expressing optoSTING (STING-mCherry-Cry2), optoFUS-N (Fus-N-mCherry-Cry2), or optomCherry (mCherry-Cry2) at the indicated times. All imaged cells had a similar protein expression level and were activated under identical conditions. For all Extended Data Fig. 2, data were representatives of at least three independent experiments. Confocal images represented at least ten cells. Scale bar, as indicated. Nuc, Nucleus. See numerical data in source data.

Source data

Extended Data Fig. 3 STING condensation requires STING IDR.

a, Human STING309-379 (top) and mammalian STING (bottom) C-terminal region (309-379), were intrinsically unstructured and conserved (https://iupred2a.elte.hu/). b, 293T cells were transfected with plasmid expressing STINGWT-GFP (bottom) or STING d309-379-GFP (top). After 12 h, cells with similar fluorescence intensity were chosen and confocal images were taken. Only in cells transfected with STINGWT plasmid, STING condensates emerged and grew larger with increased STING protein expression. Percentage of cells with STING condensates in ten fields was quantified (right, means ± SD). c, HeLa TMEM173−⁄−-STING-GFP or TMEM173−⁄−cells stably expressing STINGd309-379-GFP were treated with 2’3’-cGAMP for 3 h. Representative confocal images of STING condensates were shown. Number of condensates in each field was counted (right, mean ± SD, n = 10 fields). d, g, Quantification of integrated optical density (IOD) of condensates in Fig. 2e (d), and Fig. 2f (g), (mean ± SD, n = 2 repeats; one-way ANOVA (d), p = 0.001 (d309-379), p = 0.0013 (d309-342); unpaired t test (e), p = 0.0073). ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. 25 fields were examined and stitched for quantification as one repeat. The expression level of indicated proteins was analyzed by western blot using antibodies against GFP and GAPDH. e, STING E336 and E337 were conserved among the indicated species. f, STING disorder tendency of STING-WT309-342 and STING-E336G/E337G309-342 (http://pondr.com/). For all Extended Data Fig. 3, data were representatives of at least three independent experiments. Confocal images represented at least three independent experiments in which >95% of the cells displayed similar pattern. Scale bar, as indicated. See uncropped gel images and numerical data in source data.

Source data

Extended Data Fig. 4 STING condensation requires STING intact transmembrane topology and STING dimerization.

a, c, d, e, f, Diagram of STING transmembrane domains69 (a, top left). 293T cells were transfected with plasmid expressing STING-GFP or indicated STING truncations (a, c, d, f,). The WT, TBK1−⁄− or IRF3−⁄− HeLa cells were transfected with plasmid expressing equal amounts of STING-GFP for 18 h (e). Protein expression of the indicated STING truncation was analyzed by western blot using antibodies against GFP and GAPDH (a, c, d, f,). Representative confocal images of STING condensates were shown (a, c, d, e, f,). MOD (a) were 32.5 (WT), 31.9 (dTM1), 33.7 (d35-44), 26.5 (dTM2), 35.6 (dTM3), 25.2 (d107-116), 24.9 (dTM4), 31.5 (d107-134). MOD (d) were 44.5 (WT), 33.3 (C88S), 37.3 (C91S), 32.9 (C88S/C91S, CC/SS). MOD (e) were 65.9 (WT), 73.7 (TBK1−⁄−), 66.5 (IRF3−⁄−). MOD (f) of representative images were 94.5 (WT) and 74.4 (d371-376). Area and integrated optical density (IOD) of STING condensates per cell were quantified. 25 fields were examined and stitched for quantification as one repeat. (mean ± SD, n = 2 repeats; one-way ANOVA (a, c, d); p < 0.0001 (a, area); p = 0.0023 (dTM1), p = 0.0005 (d35-44), p = 0.0009 (dTM2), p = 0.0076 (dTM3), p = 0.0044 (d107-116), p = 0.0012 (dTM4), p = 0.0007 (d107-134) (a, IOD); p < 0.0001 (c); p > 0.05 (d, e)). b, 293T cells were transfected with plasmid expressing cGAS, STING WT or indicated mutants with an IFNβ–Luc reporter system for 24 h before a luciferase assay was performed (mean ± SD, n = 2 biologically independent samples; one-way ANOVA, p < 0.0001). For all Extended Data Fig. 4, data were representatives of at least three independent experiments. Confocal images are representatives of at least three independent experiments in which >95% of the cells displayed similar pattern. Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images and numerical data in source data.

Source data

Extended Data Fig. 5 2’3’-cGAMP initiates and promotes STING phase separation and gel-like transition.

a, The Coomassie staining (left) and western blot (right) of indicated purified proteins were shown. STING EE/GG, STING E336G/E337G. Antibodies against STING, TBK1 and IRF3 were used. b, Phase-separation diagram of STING and NaCl at indicated concentrations at 37°C in a phase separation buffer (50 mM Tris-HCl, 750 mM NaCl, 10% BSA, 5% PEG, 0.15% SLS, pH 7.5). Green dots: Phase separation; white dots: no phase separation; black dots: inaccessible conditions (purified STING protein can be homogenous at most to 3 μM in 2 M NaCl buffer at room temperature leading to some inaccessible ionic conditions). Images represented all fields in wells. c, Representative fluorescence confocal images showing fusion of STING droplets. STING phase separation was performed as in Fig. 3a (37°C). d, Representative images of STING phase separation reconstitution of different STING concentration at the physiological condition (50 mM Tris-HCl, 150 mM NaCl, 0.03% SLS, pH 7.5, 37°C). STING concentration was increased by centrifugation as in Methods. STING droplets in 0.1 μM and 1.5 μM were imaged under x100 objective while condensates in 40 μM was too large and needed to be imaged under x40 objective. e, Representative confocal images of STING droplets formed by in vitro reconstitution using purified WT or E336G/E337G mutant STING with the same protein concentration at 4°C as in Fig. 3a (4°C) for 12 h. f, Phase-separation diagram of human STING and NaCl after mixing with CDN (c-di-GMP or 2’3’-cGAMP) at indicated concentrations at 37°C in the phase separation buffer (50 mM Tris-HCl, 750 mM NaCl, 10% BSA, 5% PEG, 0.15% SLS, pH 7.5) for 10 min. Green dots: phase separation; white dots: no phase separation; red dots: gel-like transition. For all Extended Data Fig. 5, data were representatives of at least three independent experiments. Confocal images represented all fields in a well. Scale bar, as indicated. See uncropped gel images in source data.

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Extended Data Fig. 6 STING puzzle-like structures are formed via two routes.

a, A representative TEM micrograph of puzzle-like structures (b1) without surrounding membrane (b2) as in Fig. 4b. b, Full 3D-reconstructed images of Fig. 4c (b) with different views as indicated. Nucleus and mitochondria (Mit) were labeled. c, cGAS transfection-activated COS-7 cells with endogenous STING (untagged) expression level was analyzed by TEM. Images of puzzle-like structures were shown. The indicated protein expression level was analyzed by western blot using indicated antibodies. d, 293T cells were transfected with plasmid expressing cGAS and STINGd309-379-GFP. CLEM was used to trace cells expressing STINGd309-379-GFP as in Fig. 4c. Cells were stained by OTO and analyzed by TEM. Several TEM images of different parts in the same STINGd309-379-GFP expressing cell were stitched and shown. No puzzle-like structure was observed. e, 293T cells transfected with plasmid expressing cGAS and STING-GFP were imaged, fixed, stained by OTO method, and analyzed by TEM. The puzzle-like structures were developed from small tangled ER membranes. Two tangled ER membranes jointed to form the puzzle-like structures (arrows). Images represented at least five cells with the same pattern. f, Cells were treated as in (e). The zoomed images of annulate lamella structures consisted of two highly compacted ER membranes (paired arrows). g, A diagram illustrating two ways to form the puzzle-like structure. In one way, the puzzle-like structure emerged from a highly organized annulate lamella that directly transformed into puzzle-like structures, in which the central part of an annulate lamella appeared to release membranes to provide pieces and built up the puzzle-like structure gradually. In the other way, it started from very compacted ER granules that were full of small tangled ER fractions. For all Extended Data Fig. 6, data were representatives of at least three independent experiments. TEM images represented at least five cells with similar pattern. Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images in source data.

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Extended Data Fig. 7 The STING puzzle-like structure insulates STING-TBK1 from IRF3.

a, TEM images of COS-7 cells stably expressing STING-APEX. STING-APEX staining is diffused outside ER membrane in unstimulated cells. b, TEM images of puzzle-like structures coexisted with tangled ER fractions with varying diameters in 293T cells transfected with plasmid expressing cGAS and STING-APEX. c, 293T cells transfected with plasmid expressing cGAS, STING-GFP, TBK1-mCerulean and IRF3-mCherry for 24 h. d, Representative images of STING condensate reconstitution in vitro by mixing of STING (Alexa Fluor 488 labeled, 1.5 μM) with TBK1(Alexa Fluor 594 labeled, 1.5 μM) or IRF3 (Alexa Fluor 594 labeled, 1.5 μM) in buffer containing 50 mM Tris-HCl, pH 7.5, 750 mM NaCl, 0.15% SLS at 4°C for 12 h. e, COS-7 cells expressing STING-GFP were activated by cGAS transfection. Immunofluorescence using an antibody against human p-TBK1 was performed. Nucleus was stained by DAPI. f, 293T cells were transfected with the same amount of plasmid expressing cGAS, Flag-TBK1, HA-IRF3, and increasing amounts of plasmid expressing STING for 18 h. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot using indicated antibodies. g, 293T cells were transfected with the same amount of plasmid expressing cGAS, HA-TBK1, Flag-IRF3, and increasing amounts of plasmid expressing STING for 18 h. Cells lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot as indicated using antibodies against GFP and GAPDH. For all Extended Data Fig. 7, data were representatives of at least three independent experiments. TEM images represented at least five cells. For confocal images, more than 95% area of field displayed similar pattern. Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images in source data.

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Extended Data Fig. 8 The STING condensate prevents overactivation of innate immunity.

a, 293T cells were transfected with plasmid expressing cGAS and STING-mCherry-Cry2 or STING-mCherry for 24 h, followed by blue light exposure for the indicated times. Blue-light activation of indicated cells at the indicated times (minutes) were monitored. b, Cells transfected with cGAS and optoSTING were exposed to blue-light for 30 min before fixed, stained by OTO and analyzed by TEM. Puzzle-like structures were found in blue-light-activated optoSTING expressing cells. c, FRAP of optoSTING condensates in cells from (a). Quantification of FRAP was shown (mean ± SD, n = 4 optoSTING condensates). d, A model illustrating the experiment procedure of Fig. 7g (left) and a repeated experiment of STING condensate’s function in vitro (right). ① A homogenous STING solution containing the WT or E336G/E337G (EE/GG) STING was incubated in the physiological buffer (50 mM Tris, 150 mM NaCl, 0.03% SLS, pH 7.5) at 4°C overnight to induce STING condensation. WT STING formed STING droplets while EE/GG STING remained homogenous. ② Fresh WT STING without incubation, incubated E336G/E337G or WT STING was next mixed with 100 μl whole cell extracts (CEs) from hypotonic buffer-disrupted TMEM173−⁄− THP-1 cells and 5 μg/ml 2’3’-cGAMP in a reaction buffer at 37°C for 1 h. 200 ng STING protein (Input STING) for each was used for incubation. ③ The input STING and reaction mixtures were analyzed by western blot using the indicated antibodies. The repeated experiment was performed as in Fig. 7g, except that STING condensates were obtained by concentration as in Extended Data Fig. 5d. The WT STING remained homogenous at 0.75 μM of concentration, but started to condensate at 2.4 μM and the condensates grew larger at 12 μM. EE/GG STING remained homogenous regardless of concentrations. For all Extended Data Fig. 8, data were representatives of at least three independent experiments. Images represented at least ten cells with the same pattern (a, b). > 95% area of field displayed similar pattern (e). Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images and numerical data in source data.

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Extended Data Fig. 9 The STING condensate formation was affected by SAVI mutants and its gel-like transition was inhibited by microtubule inhibitors.

a, COS-7-STING-GFP cells were activated by cGAS-transfection (left three panels) or SeV (right panel) for 13 h and treated with microtubule inhibitors as in Fig. 8c. b, c, THP-1 cells were treated as in Fig. 8c. Supernatants were collected and analyzed by type I-IFN bioassay (b, mean ± SD, n = 2 biologically independent samples; two-way ANOVA, p = 0.0017 (VACV-Colc), p < 0.0001 (VACV-Vinc/Noco, HSV-1-Vinc), p = 0.0002 (VACV-Vinb), p > 0.05 (SeV-Colc, SeV-Vinc, SeV-Noco, SeV-Vinb), p = 0.0001 (HSV-Noco), p = 0.0405 (HSV-Vinb), p = 0.036 (HSV-Colc)). Cells were stained with propidium iodide (PI) and analyzed by FACS to monitor cell viability (c, mean ± SD, n = 2 biologically independent samples; one-way ANOVA, p > 0.05). d, Area and IOD of STING condensates per cell of Fig. 8d were quantified. 25 fields were examined, stitched and used for quantification as one repeat. (mean ± SD, n = 2 biologically independent samples; one-way ANOVA; For IOD, p = 0.0207 (V147L), p = 0.0003 (N154S), p = 0.0006 (V155M), p = 0.0015 (G166E), p = 0.0004 (SAVI); For area, p = 0.0357 (V147L), p = 0.0007 (N154S), p = 0.0017 (V155M), p = 0.0036 (G166E), p = 0.001 (SAVI)). e, 293T cells were transfected with plasmid expressing cGAS, STING-GFP WT or indicated mutants with an IFNβ–Luc reporter system for 24 h before a luciferase assay was performed (mean ± SD, n = 2 biologically independent samples). Indicated protein expression level was analyzed with western blot using antibodies against GFP and GAPDH. f, A model depicting the formation of the STING Phase-separator. Top left: normal ER membrane with STING protein. Top right: specified ER membrane in the STING Phase-separator with highly enriched 2’3’-cGAMP, STING and TBK1. Bottom: Intracellular 2’3’-cGAMP drives STING condensation and formation of the STING Phase-separator. For all Extended Data Fig. 9, data were representatives of at least three independent experiments. Scale bar, as indicated. See uncropped gel images and numerical data in source data.

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Supplementary information

Reporting Summary

Supplementary Video 1

HeLa TMEM173−⁄−-STING-GFP cells were infected with VACV. Fission and fusion of STING condensates were imaged. Images were taken 8 h after infection. One representative video of 10 fields of at least three independent experiments is shown.

Supplementary Video 2

HeLa TMEM173−⁄−-STING-GFP cells were treated with 2′3′-cGAMP. FRAP was performed after 45 min and images were taken. Bleached areas are labelled by white arrows. One representative video of at least three independent experiments is shown.

Supplementary Video 3

293T cells transfected with plasmid expressing STING-mCherry-Cry2 were activated by blue light to induce STING condensate formation. Induced STING condensates are labelled by white arrows. One representative video of at least three independent experiments is shown.

Supplementary Video 4

COS-7 cells were transfected with plasmids expressing cGAS and STING-mCitrine, TBK1-mCerulean and IRF3-mCherry. Live cell images were taken after 6 h. Proteins names are labelled. One representative video of 10 fields of at least three independent experiments is shown.

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Yu, X., Zhang, L., Shen, J. et al. The STING phase-separator suppresses innate immune signalling. Nat Cell Biol 23, 330–340 (2021). https://doi.org/10.1038/s41556-021-00659-0

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