Clathrin-associated AP-1 controls termination of STING signalling – Nature

Cell culture

HeLa (CCL-2) cells were obtained from Sigma-Aldrich. HEK293T cells were a gift from D. Trono, originally purchased from ATCC (SD-3515). THP-1 cells and WI-38 cells were obtained from ATCC. HaCaT cells were obtained from CLS. Primary human alveolar epithelial cells (epithelial cells) were obtained from a commercial supplier (Cell Biologics). MEFs (μ1 KO cells and μ1 KO cells reconstituted with μ1A) were a gift from P. Schu. Primary fibroblast cells from three patients with SAVI were provided by R. Goldbach-Mansky. HeLa, HEK293T, WI-38, HaCaT and primary fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, 41965039) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco SKU, 10270106), 100 IU ml⁻¹ penicillin–streptomycin (BioConcept, 4-01F00-H), 2 mM l-glutamine (Thermo Fisher Scientific, 25030024) and 1 mM sodium pyruvate (BioConcept, 5-60F00-H) at 37 °C and at atmospheric O₂ and 5% CO₂. THP-1 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, 21875091) supplemented with 10% FBS, 1× penicillin–streptomycin–l-glutamine (Corning, 30-009-Cl) and 1× 2-mercaptoethanol (Gibco) at 37 °C and at atmospheric O₂ and 5% CO₂. MEFs were cultured in DMEM (Thermo Fisher Scientific, 41965039) supplemented with 15% (v/v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco SKU, 10270106), 100 IU ml⁻¹ penicillin–streptomycin (BioConcept, 4-01F00-H) and 1 mM sodium pyruvate (BioConcept, 5-60F00-H) at 37 °C and at atmospheric O₂ and 5% CO₂. Primary human alveolar epithelial cells (epithelial cells) were cultured in complete human epithelial cell medium (Cell Biologics, H6621), according to the supplier’s instructions. Cell lines were repeatedly tested for mycoplasma by PCR. No method of cell line authentication was used.

Plasmids

For CRISPR–Cas9 plasmids, single-guide RNA (sgRNA) targeting TBK1, IRF3, cyclic GMP-AMP synthase (cGAS), AP-1σ1 and STING were designed using the web tool CRISPOR⁴⁷. sgRNAs targeting TBK1, IRF3, cGAS and AP-1σ1 were cloned in a pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene, 62988), whereas sgRNAs targeting STING were cloned in a pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene, 48138). Both plasmids were gifts from F. Zhang⁴⁸. The pEF-Bos-based STING truncations (1–341 and 1–317) and mutations (E360A, LI364/365AA, ELI360/364/365AAA, LR374/375ΑΑ, L364A, L364F and I365A) were obtained by site-directed mutagenesis. pEF-Bos-human TBK1-Flag-His was a gift from S. Cerboni. TBK1(S172A) was generated by single-amino-acid mutation. pCDNA3-HA γ-adaptin 1(AP1G1) (Addgene, 10712) was purchased from Addgene. pCDNA3-HA-AP1S1 was generated by inserting the coding sequences of AP1S1 flanked by 5’ BamHI and 3’ XhoI sites into the pCDNA3 vector. AP1G1(R15E), AP1S1(I103S) and AP1S1(V88D) were obtained by single-amino-acid mutagenesis. The primers used for plasmid constructions and sgRNA sequences are provided in Supplementary Table 1. Plasmids for NF-κB-Luc (Promega, E8491) were purchased from Promega and those for pIFNβ–GLuc were previously described⁸. All constructs were confirmed by DNA sequencing.

Stable cell lines

HeLa STING KO cells were obtained from F. Martinon⁴⁹. HeLa^STING cells and HeLa^GFP–STING cells were generated from HeLa STING KO cells by infection with a pTJ lentiviral vector carrying Flag–STING or GFP–STING, respectively, and a puromycin resistance gene. Cells were selected with puromycin (2 µg ml⁻¹). HeLa TBK1 KO cells, HeLa RIF3 KO cells and HaCaT σ1 ΚΟ cells were generated using CRISPR–Cas9 technology. In brief, HeLa cells were plated in six-well culture plates at about 80% confluency and transfected. Per well, 3.5 µl Lipofectamine 2000 (Life Technologies, 11668019) and 1 µg plasmid-DNA were each diluted in 125 µl OptiMEM (Life Technologies, 31985047), mixed, incubated for 5 min and added on top of the well. The next day, the culture medium was replaced and cells were put under puromycin (5 µg ml⁻¹) selection for three days. Surviving cells were expended in antibiotic-free medium and sorted by fluorescence-activated cell sorting (FACS) into single clones three days later. Growing clones were characterized by western blotting and selected for the absence of TBK1. HeLa cGAS/STING KO cells were generated using CRISPR–Cas9 technology. HeLa cells were transfected with a pX459-sgcGAS plasmid for 24 h and then selected with puromycin for three days. Surviving cells were expanded in antibiotic-free medium and then transfected with the pX458-sgSTING plasmid in the same way for three days and sorted by FACS. The cells expressing GFP were maintained as HeLa cGAS/STING double KO cells. Growing clones were characterized by western blotting and selected for the absence of cGAS and STING.

Transfection

For plasmid transfection, cells were transfected with plasmids and Lipofectamine 2000 reagent (Invitrogen, 11668019) (for all imaging experiments) or GeneJuice transfection reagent (Millipore, 70967) (for all other experiments) following the manufacturer’s respective protocols. For the siRNA knockdown, 3 × 10⁴ cells were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778075) and 40 pmol siRNA, following the manufacturer’s protocol, followed by three days of incubation. Medium containing transfection reagents was replaced with fresh medium 6 h after transfection. Silencer select predesigned siRNAs (4390847), siAP1G1 (s1143), siAP1B1 (s1141), siAP1S1 (s3115) and siAP1S3 (s43490) were purchased from Thermo Fisher Scientific; siIRF3 and siTBK1 were synthesized by Mircosynth. The sequences of siRNAs are provided in Supplementary Table 1.

Stimulation of cells

Cells were treated with 2.5 μM diABZI (Selleckchem, S8796) and collected at the indicated time points. For cGAMP, 90mer and IVT4 stimulation, 0.1 μM cGAMP (Invivogen), 0.2 μg 90mer or 0.5 μg IVT4 was transfected using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s protocol, and cells were incubated for 3 h. The DNA sequences of 90mer is provided in Supplementary Table 1. Poly(I:C) (Invivogen) was added to the cell medium at a final concentration of 10 μg ml⁻¹ for 24 h. After infection with the HSV-1 KOS strain (MOI of 10), infected cells were incubated for 6 h. Pretreatment with BX795 (MedChemExpress) was performed at 2 µM for 24 h and pretreatment with bafilomycin A1 (Baf A1; Sigma) was performed at 20 nM for 1 h. MEFs treated with 5 μg ml⁻¹ or 40 μg ml⁻¹ DMXAA (Invivogen) were collected 2 h or 3 h after stimulation. For STING inhibition by H-151, H-151 (2 µM) was added into cells every 24 h for three days before cells were examined by RT–qPCR.

Antibodies

Primary antibodies used: mouse monoclonal anti-vinculin (hVIN-1) (Sigma-Aldrich, V9264, immunoblot 1:5,000), rabbit monoclonal anti-GAPDH (14C10) (Cell Signaling Technology, 2118, immunoblot 1:3,000), mouse monoclonal anti-Flag (M2) (Sigma-Aldrich, F1804, immunoblot 1:3,000), rabbit monoclonal anti-human phospho-STING (Ser366) (D7C3S) (Cell Signaling Technology, 19781, immunoblot 1:3,000), rabbit monoclonal anti-phospho-TBK1/NAK (Ser172) (D52C2) (Cell Signaling Technology, 5483, immunoblot 1:1,000), rabbit monoclonal anti-phospho-IRF3 (Ser386) (EPR2346) (Abcam, ab76493, immunoblot 1:1,000), rabbit monoclonal anti-TBK1/NAK (D1B4) (Cell Signaling Technology, 3504, immunoblot 1:1,000), rabbit polyclonal anti-TMEM173/STING (Proteintech, 19851-1-AP, immunoblot 1:1,000), rabbit monoclonal anti-IRF3 (D6I4C) (Cell Signaling Technology, 11904, immunoblot 1:1,000), rabbit monoclonal anti-clathrin heavy chain (P1663) (Cell Signaling Technology, 2410, immunoblot 1:500), mouse anti-clathrin heavy chain monoclonal antibody (X22) (Thermo Fisher Scientific, MA1-065, immunofluorescence (IF) 1:100), rabbit polyclonal anti-AP1S1 (Thermo Fisher Scientific, PA5-63913, immunoblot 1:1,000), rabbit polyclonal anti-AP1G1 (Thermo Fisher Scientific, PA5-65290, immunoblot 1:1,000), rabbit polyclonal anti-AP1B1 (Sigma-Aldrich, HPA065226, immunoblot 1:1,000), rabbit polyclonal anti-AP1M1 (Proteintech, 12112-1-AP, immunoblot 1:1,000), mouse monoclonal anti-HSV-1 ICP0 (11060) (Santa Cruz, sc-53090, immunoblot 1:500), mouse monoclonal anti-HA.11 epitope tag (16B12) (Biolegend, MMS-101R, immunoblot 1:2,000). mouse monoclonal γ-adaptin (AP1G1) (100/3) (Sigma-Aldrich, A4200, IF 1:100), mouse monoclonal EEA1 (E9Q6G) (Cell Signaling, 48453, IF 1:100), mouse monoclonal LAMP1 (H4A3) (Abcam, ab25630, IF 1:100), rabbit monoclonal anti-human phospho-STING (Ser366) (D8K6H) (Cell Signaling Technology, 40818, IF 1:100, STED 1:50), mouse monoclonal RAB7 (E9O7E) (Cell Signaling Technology, 95746, IF 1:100) and sheep polyclonal human TGN46 (Bio-Rad, AHP500G, IF 1:200). HRP-conjugated secondary antibodies used: donkey anti-rabbit IgG (H+L)-HRP (Jackson ImmunoResearch, 711-036-152, immunoblot: 1:5,000) and donkey anti-mouse IgG (H+L)-HRP (Jackson ImmunoResearch, 715-036-151, immunoblot: 1:5,000). Fluorescence-conjugated secondary antibodies used: goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor 647-conjugated (Invitrogen, A-21241, IF 1:800), donkey anti-sheep IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488-conjugated (Invitrogen, A-11015, IF 1:800), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568-conjugated (Invitrogen, A-11011, IF 1:800) and goat anti-rabbit IgG F(ab) ATTO647N (H+L) (Hypermol,2318, IF 1:500). Antibody details are provided in Supplementary Table 1.

RT–qPCR

Cells were lysed in the RLT buffer (Qiagen). RNA was extracted following the manufacturer’s protocol (Qiagen RNeasy Plant Mini Kit). RNA was reverse-transcribed using the RevertAid First Strand cDNA synthesis Kit (Thermo Fisher Scientific) and analysed by RT–qPCR in triplicate or quadruplicate using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The qPCR reactions were run on a QuantStudio 7 Real-Time PCR system (Thermo Fisher Scientific). GAPDH was used as a housekeeping gene for normalization. Primer sequences are provided in Supplementary Table 1.

Western blotting and immunoprecipitation

Cells were collected, quickly rinsed with 1× phosphate-buffered saline (PBS) and lysed in lysis buffer (20 mM Tris pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 1.5 mM MgCl₂, 2 mM EGTA, 2 mM DTT and 1× cOmplete Protease Inhibitor Cocktail (Roche)) on ice for 30 min and centrifuged at 12,000 rpm, 4 °C for 10 min. Supernatants were boiled with 4× loading buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol, 0.4 M DTT, 0.4% bromophenol blue) for 10 min. Proteins were resolved by SDS–PAGE using SurePAGE precast gels (GenScript) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad) following the manufacturer’s instructions. Membranes were blocked with 2% bovine serum albumin (BSA) + 1% milk in PBST (PBS + 0.05% Tween-20) at room temperature for 1 h and then incubated with the primary antibody (diluted in PBST) at 4 °C overnight. After washing in PBST, membranes were incubated with the secondary antibody at room temperature for 1 h. Membranes were washed with PBST, visualized with western blotting detection reagent (Advansta), and imaged using the ChemiDoc XRS Bio-Rad Imager and Image Lab Software. Band intensities were quantified using Fiji software (NIH). For immunoprecipitation, cells were seeded into six-well plates and were transfected with the indicated plasmids. Sixteen hours after transfection, cells were lysed in lysis buffer on ice for 30 min and centrifuged at 12,000 rpm at 4 °C for 10 min. Supernatants were transferred into new tubes and mixed with anti-Flag M2 magnetic beads (Sigma-Aldrich, M8823) at 4 °C overnight on a rotator. After three to six washes with lysis buffer and one to two washes with cold 1× PBS, the beads were boiled with 1× loading buffer for 10 min. Samples of 20 μl were loaded into gel after a short centrifugation, and this was followed by SDS–PAGE and immunoblotting analysis.

Luciferase assay

HEK293T cells were plated into 96-well plates and transfected with non-targeting control or the combination of siRNAs targeting AP1G1, AP1S1 and AP1B1 for three days. Cells were transfected using GeneJuice transfection reagent (Millipore) with an IFNβ promoter–reporter plasmid (pIFNβ-GLuc) in combination with a STING-expressing plasmid (pEF-Bos-Flag-STING). Sixteen hours after transfection, cells were stimulated with fresh medium containing 2.5 μM diABZI for 6 h. Gaussia luciferase activity was measured in the supernatants using coelenterazine (PJK GmbH) as substrate. For the measurement of NF-κB promoter luciferase activity, cells were transfected with siRNAs as described before and then transfected using GeneJuice with a NF-κB promoter–reporter plasmid (NF-κB-Luc). After 16 h, cells were stimulated with fresh medium containing 2.5 μM diABZI for 18 h. The promoter activity was determined using the Bright-Glo Luciferase Assay System (Promega). The expression of proteins was confirmed by immunoblotting. For determining the number of viable cells in culture, cells were seeded into a 96-well plate and were measured with CellTiter- Glo Luminescent Cell Viability Assay System (Promega) every 24 h following the producer’s instructions.

CCV extraction

CCV extraction was performed according to a previously described protocol⁵⁰. In brief, cells from one confluent 500-cm² dish were treated with 2.5 μM diABZI for 2 h and then rinsed with PBS twice. Cells were scraped into 5 ml buffer A (0.1 M MES, pH 6.5, 0.2 mM EGTA, 0.5 mM MgCl₂) and homogenized by pipetting up and down more than 25 times using a 5-ml syringe with a 22-G needle attached. Cell lysates were centrifuged at 4,100g, 4 °C for 32 min. Supernatants were moved into new tubes and treated with 50 μg ml⁻¹ ribonuclease A on ice for 30 min followed by centrifugation at 50,000 rpm, 4 °C for 30 min using a Type 70 Ti rotor (Beckman Coulter). Pellets were resuspended in 300 μl buffer A and mixed with an equal volume of buffer B (12.5% (w/v) Ficoll, 12.5% (w/v) sucrose in buffer A), followed by centrifugation at 20,000 rpm, 4 °C for 25 min. Supernatants were transferred into new tubes and diluted with four volumes of buffer A, and centrifuged at 40,000 rpm, 4 °C for 30 min to obtain the CCV-enriched fraction.

Sample preparation for confocal microscopy

HeLa^STING cells were plated in CellCarrier-96 Ultra Microplates (PerkinElmer, 6055302) at a density of 10,000 cells per well and left for at least 5 h to adhere. Cells were then stimulated by adding diABZI at 1 µM (MedChemExpress, HY-103665) for the indicated amount of time. In time-course experiments, cells were stimulated in a sequential manner and fixed all at the same time. At the end of the stimulation, the wells were washed once with PBS, and then cells were fixed by adding paraformaldehyde 4% in CBS buffer (10 mM MES pH 6.9, 138 mM KCl, 2 mM MgCl₂ and 2 mM EGTA) for 5–10 min at room temperature. Cells were then washed three times for at least 5 min in PBS before blocking for 1–2 h at room temperature with PBS supplemented with 0.1% (v/v) saponin and 5% (v/v) heat-inactivated FBS and later incubating overnight at 4 °C with the primary antibodies diluted in staining solution (PBS supplemented with 0.1% (v/v) saponin) and 1% (w/v) BSA (Sigma-Aldrich, A7906)). Antibodies and concentrations are listed in Supplementary Table 2. The cells were then washed with PBS three times for 5 min and incubated for 1.5 h at room temperature in secondary antibodies diluted in staining solution. From then on, the plate was protected from light. Cells were washed twice more (5 min each) in PBS and incubated for 30–60 min in Hoechst 33342 (Sigma-Aldrich, B2261) 0.2 µg ml⁻¹ in PBS. Cells were then put back into 100 µl PBS per well and either imaged directly or kept at 4 °C until imaging. For experiments with KD of AP-1, HeLa^STING cells were plated (60,000 cells per well in a six-well plate) and leftt to adhere for 6–16 h. They were then transfected with siRNAs using Lipofectamine RNAiMax reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Silencer select predesigned siRNAs were purchased from Thermo Fisher Scientific: negative control (4390847), or siAP1G1 (s1143), siAP1B1 (s1141) and siAP1S1 (s3115); see details in Supplementary Table 1. A total of 2 µl at 10 µM of siRNAs (equally split between the three siRNAs for the siAP-1 wells) combined with 7 µl of lipofectamine RNAiMax reagent in 400 µl OptiMEM total was used per well. The medium was changed after 6–16 h. Three days after siRNA treatment, cells were replated into microscopy plates (10,000 cells per well) and the experiments were continued as described above for non-siRNA-treated cells.

Imaging and analysis for confocal microscopy

Fixed and stained 96-well plates were then imaged on two microscopes: a confocal Leica SP8 inverted microscope equipped with an HC PL APO 63×/1.40/oil (magnification/N.A./immersion) objective and HyD detectors, operated with the Leica LAS X software; and a PerkinElmer Operetta CLS operated with the PerkinElmer Harmony software and equipped with an Andor Zyla 5.5 camera, and with an LD C Apochromat objective with magnification/N.A./immersion of 63×/1.4/water. Image analysis and quantification were performed by combining PerkinElmer Harmony (v.4.9) and Fiji (v.2.3.0), and data were further processed with KNIME (v.4.3.2) and GraphPad PRISM 9 (v.9.3.1). The panels of images were assembled using OMERO (v.5.11.0)⁵¹. In more detail, all depicted cell images were acquired with the Leica SP8 confocal microscope (63×), except for the images confirming that pSTING still accumulates at the TGN after treatment with siRNA against AP-1 (compared to siNC), which are images captured on the Operetta (63×). Quantifications of the total area of pSTING or AP1G1 intensity were calculated in Harmony software on the basis of Operetta (63×) images.

Airyscan microscopy

HeLa^STING cells were plated in µ-Slide eight-well chamber slides (ibidi, 80826-IBI) at a density of 10,000 cells per well and left to adhere overnight. Cells were then stimulated by adding diABZI at 1 µM (MedChemExpress, HY-103665) for 0, 150 or 360 min (Fig. 1b depicts the 150-min time point and Extended Data Fig. 1e the 0- and 360-min ones) or only 0 and 150 min (Fig. 2a and Extended Data Fig. 1c,d). In time-course experiments, cells were stimulated in a sequential manner and fixed all at the same time. At the end of the stimulation, the wells were washed once with PBS, and then cells were fixed by adding paraformaldehyde 4% in CBS buffer (10 mM MES pH 6.9, 138 mM KCl, 2 mM MgCl₂, 2 mM EGTA) for 5–10 min at room temperature. Cells were then washed three times for at least 5 min in PBS before blocking for 1–2 h at room temperature with PBS supplemented with 0.1% (v/v) saponin and 5% (v/v) heat-inactivated FBS and later incubating overnight at 4 °C with the primary antibodies diluted in staining solution (PBS supplemented with 0.1% (v/v) saponin) and 1% (w/v) BSA (Sigma-Aldrich, A7906)). Antibodies and concentrations are listed in Supplementary Table 2. The cells were then washed with PBS three times for 5 min and incubated for 1.5 h at room temperature in secondary antibodies diluted in staining solution. From then on, the plate was protected from light. Cells were washed twice more (5 min each) in PBS and incubated for 30–60 min in Hoechst 33342 (Sigma-Aldrich, B2261, blue on the depicted images) 0.2 µg ml⁻¹ in PBS. Cells were then put back into 200 µl PBS per well and either imaged directly or kept at 4 °C until imaging. Imaging was performed with a Zeiss LSM 980 Inverted microscope (multi-purpose confocal with 32 Channels AiryScan, tPMT, widefield and bright-field capability) using the Plan-Apochromat 63×/1.40/oil (magnification/N.A./immersion) objective and the AiryScan mode and images were processed in the ZEN software using embedded AiryScan processing (3D-mode and ‘Normal’ resolution). Image analysis and quantifications were performed with Fiji (v.2.3.0). Colocalization analysis were performed on Fiji using the BIOP JACoP plug-in with Otsu thresholding for all channels. Data were further processed with KNIME (v.4.3.2) to combine them and remove cells with threshold values lower than 500 for pSTING (considered background), and the results were then plotted with GraphPad PRISM 9 (v.9.3.1). The panels of images were assembled using OMERO (v.5.11.0) (ref. ⁵¹).

CLEM

HeLa^GFP–STING cells were plated in glass-bottomed Petri dishes (MatTek, P35G-1.5-14-CGRD) with an alpha-numeric grid pattern at a density of 100,000 cells per dish and left to adhere overnight. They were stimulated by adding diABZI at 1 µM (MedChemExpress, HY-103665) for 2.5 h. They were then chemically fixed with a buffered solution of 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The dishes were then screened with light microscopy to identify cells of interest, which were imaged with both transmitted and fluorescence microscopy (AiryScan mode as described above) to record their position on the grid. The cells were then washed thoroughly with cacodylate buffer (0.1 M, pH 7.4), and post-fixed for 40 min in 1.0% osmium tetroxide with 1.5% potassium ferrocyanide and then 40 min in 1.0% osmium tetroxide alone. They were finally stained for 40 min in 1% uranyl acetate in water before being dehydrated through increasing concentrations of alcohol and then embedded in Durcupan ACM resin (Fluka). The dishes were then filled with 1 mm of resin and this was hardened for 18 h in a 65 °C oven. Cells of interest were then identified according to their position on the alpha-numeric grid, cut away from the rest of the material and glued to a blank resin block. Ultra-thin (50-nm-thick) serial sections were cut through the entire cell with a diamond knife (Diatome) and ultramicrotome (Leica Microsystems, UC7), and collected onto single slot grids with a pioloform support film. These sections were further contrasted with lead citrate and uranyl acetate and images taken in a transmission electron microscope (FEI Company, Tecnai Spirit) with a digital camera (FEI Company, Eagle). To correlate the light microscopy images with the electron microscopy images and identify the exact position of the Centrin-1:GFP foci, fluorescent images were overlaid onto the electron micrographs of the same cell using Photoshop (Adobe).

STED

HeLa cGAS/STING double KO cells were plated in 3.5-cm glass-bottom Petri dishes (FluoroDish, WFD35-100) at a density of 100,000 cells per dish and left to adhere overnight. They were stimulated by adding diABZI at 1 µM (MedChemExpress, HY-103665) for 2.5 h. They were then transfected with plasmids containing Flag-hSTING and mCherry-clathrin (both in pEF-Bos mammalian expression plasmid) using Lipofectamine 2000 Reagent (Invitrogen, 11668019) according to the manufacturer’s instructions. One microgram per plasmid and 4.5 µl of lipofectamine in 250 µl of OptiMEM (Life Technologies, 31985047) were used for one dish. The medium was replaced with fresh culture medium six hours after transfection. The next day, the cells were stimulated by adding diABZI at 1 µM (MedChemExpress, HY-103665) for 2.5 h. They were then fixed and stained for pSTING as described for the AiryScan microscopy samples. STED images were acquired using a Leica SP8 STED 3X (Leica Microsystems) equipped with a pulsed white light laser (WLL) as an excitation source and a 775-nm pulsed laser as a depletion light source both for mCherry and ATTO647N. Samples were imaged with a 100× objective (Leica, HC APO CS2 100×/1.40/oil) using the LAS X software (Leica Microsystems). For excitation of the respective channels, the WLL was set to 587 nm for mCherry and 647 nm for ATTO647N. Hybrid spectral detectors were used to acquire the images with a final pixel size of 9.2 × 9.2 nm. The detector time gates were set to 1.5–7.5 ns for mCherry and 0.5–6 ns for ATTO647N. Images were acquired as single planes of 1,392 × 1,392 pixels, 600 lines per second, 32× line averaging for mCherry and 16× line averaging for ATTO647N. Deconvolution of STED images was done with Huygens Remote Manager v.3.7, using Good’s roughness maximum likelihood estimation with 60 iterations and a signal-to-noise ratio equal to 2 until it reached a quality threshold of 0.03.

Protein expression and purification

6His-TEVsite-Hs ARF1 (17-181)-Q71L in pHis2 vector was expressed in BL21 (DE3) bacteria (Sigma-Aldrich, CMC0014). A single colony was inoculated in a culture flask with 100 ml LB with Ampicillin (100 µg ml⁻¹) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as preculture. Large-scale expression of the protein was started the next day by pouring 100 ml of preculture in a 5-l Erlenmeyer flask containing 2 l LB with ampicillin (100 µg ml⁻¹). The cells were grown until the optical density at 600 nm reached 0.7. Expression was then induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM while transferring the culture to an 18 °C shaking incubator overnight. The bacteria were then collected by centrifugation, solubilized in HisTrap buffer A (20 mM HEPES, 500 mM NaCl, 20  M imidazole, 1 mM DTT and 5% glycerol, pH 7.5) supplemented with 4-(2-aminoethyl)-benzolsulfonylfluoride-hydrochloride (AEBSF) and cOmplete protease inhibitors (Roche), lysed by sonication, cleared by centrifugation at 20,000g and then passed through a 5-ml nickel immobilized metal-affinity chromatography column (Cytiva, HisTrap HP, 17524802) on an fast protein liquid chromatography (FPLC) system. The protein of interest was eluted with buffer B (20 mM HEPES, 500 mM NaCl, 500 mM imidazole, 1 mM DTT and 5% glycerol, pH 7.5). ARF1 was then purified by size-exclusion chromatography through a Superdex 75 Hiload 16/600 column (Cytiva 28-9893-33). The cDNA of human LBD-STING (139–379) was cloned into a pET-28 vector with an N-terminal His6-SUMO tag. LBD-STING was expressed in Escherichia coli BL21 (DE3) with 0.4 mM IPTG induction overnight at 16 °C. The cell pellet was lysed by sonication and purified on a Ni-NTA column in 50 mM Tris at pH 8.0, 350 mM NaCl, 20 mM imidazole and 0.5 mM phenylmethanesulfonyl fluoride (PMSF). The protein was eluted with buffer containing 50 mM Tris at pH 8.0, 350 mM NaCl and 300 mM imidazole, and then loaded onto a Superdex 75 HiLoad 16/600 column (Cytiva 28-9893-33) in PBS. The sample fractions were pooled and proteins were quantified by molar absorption measurements. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing. The mutant STING proteins were expressed and purified in the same way as the wild-type STING. His-tagged AP-1β 1–584, GST-tagged AP-1γ 1–595, AP-1μ 1–423 and AP-1σ 1–154 were cloned into a pST44 vector and referred to as AP-1 core. His-tagged AP-1β 1–584, GST-tagged AP-1γ 1–595, AP-1μ 1–142 and AP-1σ 1–154 were cloned into a pST44 vector and referred to as AP-1 ΔμCTD. The AP-1 core complex in the pST44 vector was expressed in BL21 (DE3) (Sigma-Aldrich, CMC0014). A single colony was inoculated in a culture flask with 400 ml LB with ampicillin (100 µg ml⁻¹) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as preculture. Large-scale expression of the complex (8 l in total: 2 l in four 5-l Erlenmeyer flasks) was started the next day by pouring 100 ml of preculture into 2 l of Auto Induction Media Terrific Broth (Formedium, AIMTB0210) with ampicillin (100 µg ml⁻¹). Flasks were incubated with shaking at 37 °C for 6 h, then incubated at 18 °C overnight. The cells were then collected by centrifugation (4,000g, 15 min). A cell pellet of 2 l expression culture was transferred in a Falcon 50-ml tube. The 2-l expression cells were solubilized in PBS with 1 mM DTT, 1 mM EDTA and 2% glycerol at pH 7.5, supplemented with AEBSF and cOmplete protease inhibitors, then lysed by sonication. The cell lysate was clarified by centrifugation followed by 0.45-µm filtration. The supernatant was first purified on a glutathione sepharose 4B 5-ml column (Cytiva 28401748) on a FPLC system (Cytiva Aktä Pure). After TEV cleavage at 4 °C overnight in a 3,500 molecular weight cut-off dialysis tubing against PBS with 5% glycerol at pH 7.5, the sample was passed through a Superose 6 HiLoad 16/600 size-exclusion chromatography column (Cytiva 29323952) equilibrated in PBS with 2% glycerol at pH 7.5, to remove the TEV protease as well as free GST. When used for GST pull-down, TEV cleavage was skipped and the pool of GST-AP-1 eluate was directly loaded on the size-exclusion chromatography column. AP-1 ΔμCTD was expressed and purified in the same way as AP-1 core.

In vitro phosphorylation of SUMO–STING 139–379 by TBK1

The recombinant SUMO–STING stock protein was diluted in assay buffer containing 20 mM Tris, 25 mM MgCl₂, 2 mM EDTA, 4 mM EGTA and 1 mM DTT at pH 7.5, supplemented with phosphatase inhibitor cocktails and protease inhibitors. The pH was controlled before and after addition in the sample of 10 mM ATP. TBK1 (MRC PPU Reagents, DU12469) was added at a ratio of 1:20 (w/w) TBK1:STING. The reaction was performed at 4 °C overnight. The sample was loaded on a Superdex 200 Increase 10/300 GL size-exclusion chromatography column (Cytiva, 28990944) to purify phosphorylated SUMO–STING from the other reagents. The phosphorylation assay was monitored by liquid chromatography electrospray ionization mass spectrometry(LC/ESI-MS).

GST pull-down assay

For AP-1–ARF1 pull-down, 30 μg GST-tagged AP-1 complex, 10 μg ARF1 and 10 μg LBD-STING were mixed together or individually in 40 μl pull-down buffer (PBS supplemented with 2 mM MgCl₂, 1 mM GTP and 2 mM TCEP). The mixture was incubated overnight on ice. Thirty microlitres of glutathione sepharose beads (Cytiva) were incubated with the mixture for 30 min at 4 °C. Excess proteins were washed off the beads using 200 μl pull-down buffer each time for four times. Twenty microlitres of 5× SDS loading buffer was added to the resin and the mixture was boiled for 5 min. The samples were then centrifuged briefly. Five microlitres of supernatant was analysed by SDS–PAGE. The protein bands were visualized by Coomassie blue staining. For AP-1 ΔμCTD pull-down, 30 μg GST-tagged AP-1 ΔμCTD complex and 10 μg LBD-STING were mixed together or individually in 40 μl PBS supplemented with 2 mM TCEP. The mixture was incubated overnight on ice. Thirty microlitres of glutathione sepharose beads (Cytiva) was incubated with the mixture for 30 min at 4 °C. Excess proteins were washed off the beads using 200 μl PBS each time for four times. Twenty microlitres of 5× SDS loading buffer was added to the resin and the mixture was boiled for 5 min. The samples were then centrifuged briefly. Five microlitres of supernatant was analysed by SDS–PAGE. The protein bands were visualized by Coomassie blue staining.

Bio-layer interferometry

Bio-layer interferometry analyses were performed at 25 °C using a GatorPrime biosensor system (GatorBio) with streptavidin probes (GatorBio, 160002). STING, STING mutants or pSTING were mixed with biotin (EZ-Link-NHS-LC-LC-Biotin, Thermo Fisher Scientific) at a molar ratio of three biotin molecules to one STING and incubated at room temperature for 30 min, and then excess biotin was removed by using a desalting column (PD 10, Cytiva). Biotinylated STING (10 μg ml⁻¹) was immobilized onto the streptavidin biosensor (GatorBio, 18-5019) for 1 min. The tips were washed with PBS buffer for 2 min to obtain a baseline reading, then the biosensors were dipped into wells containing the various concentrations of AP-1 ΔμCTD or its mutant for 5 min, which was followed by a 10-min buffer wash to allow the dissociation of molecules from the sensor. Data analysis was performed with GraphPad PRISM 9 using a standard 1:1 binding model. Two independent experiments were performed for each sample.

Cryo-EM data acquisition

Two milligrams of GST-tag cleaved AP-1 core complex was incubated with 2 mg ARF1 for 30 min at room temperature in PBS supplemented with 2 mM MgCl₂, 1 mM GTP and 2 mM TCEP. Two milligrams of pSTING was then added, and the mixture was incubated on ice overnight. Excess ARF1 and pSTING were removed with a Superose 6 increase 10/300 GL column (Cytiva) in PBS. The AP-1–ARF1–pSTING complex fraction was collected and concentrated to 0.8 mg ml⁻¹. Aliquots of 3 μl of AP-1–ARF1–pSTING complexes were loaded onto glow-discharged holey carbon grids (Electron Microscopy Sciences, Q250AR1.3, Quantifoil, Au, R 1.2/1.3, 300 mesh). Grids were blotted for 4 s and plunge-frozen in liquid ethane using a Vitrobot at 4 °C and with 100% humidity. Grids were screened for particle presence and ice quality on a TFS Glacios microscope (200 kV), and the best grids were transferred to TFS Titan Krios G4. Cryo-EM data were collected using a TFS Titan Krios G4 transmission electron microscope (TEM), equipped with a Cold-FEG on a Falcon IV detector in electron counting mode. Falcon IV gain references were collected just before data collection. Data were collected with TFS EPU v.2.12.1 using aberration-free image shift protocol (AFIS), recording eight micrographs per ice hole. Movies were recorded at a magnification of 270,000×, corresponding to the 0.45 Å pixel size at the specimen level, with defocus values ranging from −0.8 to −1.8 μm. Exposures were adjusted automatically to 60 e⁻  Å⁻² total dose, resulting in an exposure time of approximately 3 s per movie. In total, 30,004 micrographs in EER format were collected.

Cryo-EM data processing

Motion correction was performed on raw stacks without binning using the cryoSPARC implementation of motion correction. A total of 1,701,051 particles were template-based automatically picked and particles were binned by a factor of 4. Two rounds of two-dimensional (2D) classification were performed, resulting in a particle set of 539,684 particles. Two-dimensional classification of particles showed that the relative orientation between STING and AP-1 was highly variable. Selected particles resulting from the 2D classification were used for ab initio reconstruction. After two rounds of ab initio reconstruction, 326,772 particles were selected on the basis of STING densities. The particles were re-centred and re-extracted by a binning factor of 2. The particles were subjected to iterative CTF refinement and non-uniform refinement in cryoSPARC to 2.34 Å. The reported resolutions are based on the gold-standard Fourier shell correlation 0.143 criterion. Local-resolution variations were estimated using cryoSPARC.

Model building and refinement

The AP-1–ARF1–pSTING model was generated using a published AP-1–ARF1–tetherin Nef structure (PDB 6DFF). The AP-1–ARF1 model after removing the tetherin Nef ligand was docked into the cryo-EM map in Chimera and fine-tuned by manual adjustment with Coot. The pSTING tail was docked against the cryo-EM map in Coot and the whole model was refined in PHENIX. Several loop regions of AP-1, ARF1 and pSTING were manually adjusted to fit into the map using Coot. The model was refined in real space again in PHENIX. All structure figures were made using UCSF Chimera, UCSF ChimeraX and PyMOL.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.