Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation

Abstract

The NLRP3 inflammasome, which has been linked to human inflammatory diseases, is activated by diverse stimuli. How these stimuli activate NLRP3 is unknown. Here we show that different NLRP3 stimuli lead to disassembly of the trans-Golgi network (TGN). NLRP3 is recruited to the dispersed TGN (dTGN) through ionic bonding between its conserved polybasic region and negatively charged phosphatidylinositol-4-phosphate (PtdIns4P) on the dTGN. The dTGN then serves as a scaffold for NLRP3 aggregation into multiple puncta, leading to polymerization of the adaptor protein ASC, thereby activating the downstream signalling cascade. Disruption of the interaction between NLRP3 and PtdIns4P on the dTGN blocked NLRP3 aggregation and downstream signalling. These results indicate that recruitment of NLRP3 to dTGN is an early and common cellular event that leads to NLRP3 aggregation and activation in response to diverse stimuli.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: NLRP3 forms multiple puncta after stimulation.
Fig. 2: NLRP3 aggregates on dTGN.
Fig. 3: NLRP3 is recruited to dTGN via its polybasic region.
Fig. 4: NLRP3 is recruited to dTGN via binding to PtdIns4P.
Fig. 5: NLRP3 binding to PtdIns4P is essential for inflammasome activation.
Fig. 6: Binding to PtdIns4P on dTGN is essential for K+-efflux-independent NLRP3 activation.

Data availability

All important data generated or analysed during this study are included in this article. Additional supplementary data are available from the corresponding author upon request.

References

  1. Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).

    CAS  Article  Google Scholar 

  2. Lamkanfi, M. & Dixit, V. M. Inflammasomes and their roles in health and disease. Annu. Rev. Cell Dev. Biol. 28, 137–161 (2012).

    CAS  Article  Google Scholar 

  3. Bryan, N. B., Dorfleutner, A., Rojanasakul, Y. & Stehlik, C. Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J. Immunol. 182, 3173–3182 (2009).

    CAS  Article  Google Scholar 

  4. Lu, A. et al. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156, 1193–1206 (2014).

    CAS  Article  Google Scholar 

  5. Cai, X. et al. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156, 1207–1222 (2014).

    CAS  Article  Google Scholar 

  6. Rossjohn, J. et al. Structures of perfringolysin O suggest a pathway for activation of cholesterol-dependent cytolysins. J. Mol. Biol. 367, 1227–1236 (2007).

    CAS  Article  Google Scholar 

  7. Gross, O. Measuring the inflammasome. Methods Mol. Biol. 844, 199–222 (2012).

    Article  Google Scholar 

  8. Meng, G., Zhang, F., Fuss, I., Kitani, A. & Strober, W. A mutation in the Nlrp3 gene causing inflammasome hyperactivation potentiates Th17 cell-dominant immune responses. Immunity 30, 860–874 (2009).

    CAS  Article  Google Scholar 

  9. Brydges, S. D. et al. Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity. Immunity 30, 875–887 (2009).

    CAS  Article  Google Scholar 

  10. Juliana, C. et al. Non-transcriptional priming and deubiquitination regulate NLRP3 inflammasome activation. J. Biol. Chem. 287, 36617–36622 (2012).

    CAS  Article  Google Scholar 

  11. Bauernfeind, F. et al. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J. Immunol. 187, 613–617 (2011).

    CAS  Article  Google Scholar 

  12. Heo, W. D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006).

    ADS  CAS  Article  Google Scholar 

  13. Szentpetery, Z., Varnai, P. & Balla, T. Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling. Proc. Natl Acad. Sci. USA 107, 8225–8230 (2010).

    ADS  CAS  Article  Google Scholar 

  14. Hsu, F., Hu, F. & Mao, Y. Spatiotemporal control of phosphatidylinositol 4-phosphate by Sac2 regulates endocytic recycling. J. Cell Biol. 209, 97–110 (2015).

    CAS  Article  Google Scholar 

  15. Balla, T. & Varnai, P. Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci. STKE 2002, pl3 (2002).

    Google Scholar 

  16. Traub, L. M., Ostrom, J. A. & Kornfeld, S. Biochemical dissection of AP-1 recruitment onto Golgi membranes. J. Cell Biol. 123, 561–573 (1993).

    CAS  Article  Google Scholar 

  17. Boman, A. L., Zhang, C., Zhu, X. & Kahn, R. A. A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. Mol. Biol. Cell 11, 1241–1255 (2000).

    CAS  Article  Google Scholar 

  18. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).

    ADS  Article  Google Scholar 

  19. Levine, T. P. & Munro, S. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695–704 (2002).

    CAS  Article  Google Scholar 

  20. Munoz-Planillo, R. et al. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38, 1142–1153 (2013).

    CAS  Article  Google Scholar 

  21. Kanneganti, T. D. et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233–236 (2006).

    ADS  CAS  Article  Google Scholar 

  22. Gross, C. J. et al. K+ efflux-independent NLRP3 inflammasome activation by small molecules targeting mitochondria. Immunity 45, 761–773 (2016).

    CAS  Article  Google Scholar 

  23. Bonardi, V., Cherkis, K., Nishimura, M. T. & Dangl, J. L. A new eye on NLR proteins: focused on clarity or diffused by complexity? Curr. Opin. Immunol. 24, 41–50 (2012).

    CAS  Article  Google Scholar 

  24. Khan, M., Subramaniam, R. & Desveaux, D. Of guards, decoys, baits and traps: pathogen perception in plants by type III effector sensors. Curr. Opin. Microbiol. 29, 49–55 (2016).

    CAS  Article  Google Scholar 

  25. Bauernfeind, F. G. et al. Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 183, 787–791 (2009).

    CAS  Article  Google Scholar 

  26. Tanaka, Y. & Chen, Z. J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci. Signal. 5, ra20 (2012).

    Article  Google Scholar 

  27. Fernandes-Alnemri, T., Yu, J. W., Datta, P., Wu, J. & Alnemri, E. S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009).

    ADS  CAS  Article  Google Scholar 

  28. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. Balla and M. Seaman for reagents, C. Pasare for sharing bones from ASC-deficient mice, B. Beutler for NLRP3-deficient mice, Molecular Biology Imaging Facility, Electron Microscopy Core Facility and O’Brien Kidney Research Center at UT Southwestern Medical Center for technical assistance, and all members of the Chen laboratory for their help and support. This work was supported by grants from the Welch Foundation (I-1389) and the Cancer Prevention and Research Institute of Texas (RP150498 and RP110430). Z.J.C. is a Howard Hughes Medical Institute Investigator.

Reviewer information

Nature thanks J. Vince and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations

Authors

Contributions

J.C. designed the study under the guidance of Z.J.C., performed all the experiments, analysed the data and prepared the manuscript. Z.J.C. designed and supervised this study and revised the manuscript.

Corresponding author

Correspondence to Zhijian J. Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 NLRP3 forms multiple puncta to activate the inflammasome pathway.

a, Endogenous NLRP3, ASC and caspase-1 are not detectable in HEK-293T cells. Extracts from HEK-293T cell lines stably expressing the indicated proteins were examined by immunoblotting. *, non-specific band. b, Reconstitution of NLRP3 inflammasome pathway in HEK-293T cells. Cells stably expressing mouse NLRP3, ASC and caspase-1 were treated with nigericin (Nig) (10 μM) for the indicated time, followed by immunoblotting. c, Highly purified NLRP3 showed signal-dependent activity in the in vitro assay. NLRP3–GFP was purified by fractionation and immunoprecipitation as detailed in Methods. Purity and activity were examined by silver staining (left) and the in vitro assay (right), respectively. d, The in vitro assay detects activation of endogenous NLRP3. RAW 264.7 cells were treated with LPS (50 ng ml−1) for 3 h and stimulated with nigericin (10 μM) for 60 min before cell extracts were collected for the in vitro NLRP3 activity assay. e, Human NLRP3 also formed puncta in response to nigericin. HeLa cells stably expressing Flag–NLRP3 were treated with nigericin (10 μM) for 80 min before immunostaining with a Flag antibody. f, NLRP3 formed multiple puncta in the absence of ASC, and formed a large speck in the presence of ASC. Cells stably expressing the indicated proteins were treated as in e before imaging. g, NLRP3 puncta possessed high activity. Left, NLRP3 puncta induced by nigericin (10 μM, 60 min) remained in the cells after saponin treatment. Nu, nucleus. Right, cell extracts with or without saponin treatment were examined by the in vitro assay. The p10 level in lane 4 is approximately 6.5 fold that in lane 6 based on quantification in imageJ (normalization by NLRP3 band intensity). Only activator cell extracts were used for tubulin immunoblot. h, Constitutively active mutants of NLRP3 formed puncta without stimulation. Cells stably expressing the indicated proteins were imaged in the absence of stimulation.

Extended Data Fig. 2 NLRP3 aggregates on stimulus-triggered dTGN.

a, Nigericin treatment induced formation of giant vesicles in the perinuclear region. HeLa cells expressing NLRP3–GFP treated with nigericin (10 μM) for 80 min were examined with phase-contrast microscopy. Mag, magnification. Cells with giant vesicle formation was quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). N.D., not detectable. b, Ultrastructural analysis of nigericin-induced dispersed TGN (dTGN) vesicles. HeLa cells treated as in a were examined by transmission electron microscopy. Blue arrowheads indicate Golgi stacks under resting conditions and red arrowheads indicate nigericin-induced dTGN vesicles. Representative images from two independent experiments (more than 30 cells were examined for each condition in each experiment) are shown. c, Nigericin triggered the formation of dTGN, on which NLRP3 aggregated. HeLa cells stably expressing the indicated protein were stimulated as in a before immunostaining for the TGN markers TGN38 and GOLGA4. d, e, cis and medial Golgi remained intact after nigericin treatment. Cells treated as in a were immunostained for GM130 (cis-Golgi marker) or giantin (cis- and medial-Golgi marker). f, NLRP3 aggregated on dispersed TGN38-positive vesicles, some of which were also EEA1-positive. HeLa cells stably expressing NLRP3–GFP and EEA1–HA were treated as in a before immunostaining for TGN38 and HA. g, ATP stimulation led to NLRP3 aggregation on dTGN. HeLa cells stably expressing NLRP3–GFP and P2X7–HA were treated with ATP (5 mM) for 80 min before imaging. P2X7 is a purinergic receptor that is essential for ATP-mediated NLRP3 inflammasome activation. h, Stimulation with gramicidin led to NLRP3 aggregation on dTGN. HeLa cells were treated with gramicidin (5 μM) for 80 min before imaging. i, DNA stimulation does not cause TGN dispersion or AIM2 recruitment to TGN. HeLa cells stably expressing AIM2–Flag were mock-transfected, transfected with poly(dA:dT) (1.5 μg ml−1) for 3 h or incubated with nigericin (10 μM) for 80 min before immunostaining with antibodies against Flag (AIM2–Flag) or TGN38.

Extended Data Fig. 3 Endogenous NLRP3 is recruited to dTGN in primary macrophages.

a, Substantial TGN disassembly occurred at early time points in wild-type BMDMs. Cells were primed with LPS (50 ng ml−1) for 3 h, followed by stimulation with nigericin (10 μM) or ATP (5 mM) for the indicated time, and immunostained for TGN38. Right, to quantify TGN disassembly, the numbers of TGN structures not connected with each other for each cell were quantified from 100 randomly selected cells and grouped as shown. b, c, Nigericin-induced cleavage of caspase-1 and IL-1β did not occur until 15 min (for nigericin) or 10 min (for ATP) after stimulation in wild-type BMDMs. Cells were treated as in a before lysates were collected for immunoblotting. d, Endogenous NLRP3 aggregation on dTGN could be detected as early as 10 min after nigericin treatment in ASC-deficient BMDMs. Cells were primed with LPS (50 ng ml−1) for 3 h, followed by nigericin (10 μM) treatment for 0, 10 or 30 min before imaging. Cells with NLRP3 puncta on dTGN were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test).

Extended Data Fig. 4 NLRP3 activity is strongly associated with dTGN but not mitochondria.

a, NLRP3 did not translocate to mitochondria upon stimulation in HeLa cells. HeLa cells expressing NLRP3–GFP were stimulated with nigericin (10 μM) or gramicidin (5 μM) for 80 min and were immunostained for TOM20 (mitochondrial marker). b, Neither nigericin- nor ATP-induced NLRP3 puncta were co-localized with mitochondria in ASC-deficient BMDMs. Cells were primed with LPS (50 ng ml−1) for 3 h, followed by nigericin (10 μM) or ATP (5 mM) treatment for 60 min before immunostaining for endogenous NLRP3 and TOM20. c, NLRP3 activity in P100 (light membrane) fraction was strongly associated with dTGN but not mitochondria. P100 fraction collected from Fig. 1c was separated by sucrose gradient ultracentrifugation. Fractions were collected and tested for activity in the in vitro NLRP3 activity assay (top). TOM20 (mitochondrial marker) and GM130 (cis-Golgi marker) were not detectable on immunoblots even after prolonged exposure. d, dTGN-localized NLRP3 puncta can initiate aggregation of ASC-PYD. HeLa cells stably expressing the indicated proteins were incubated with nigericin (10 μM) for 80 min before imaging. ASC-PYD, residues 1–90 of mouse ASC. Right, top to bottom: reconstituted z-stack image of a representative nigericin-treated cell; nigericin-treated cell co-immunostained for TGN38 (pseudocoloured in magenta); cells with ASC-PYD filaments originating from dTGN-localized NLRP3 puncta were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test).

Extended Data Fig. 5 The polybasic region mediates dTGN recruitment and activation of NLRP3.

a, NLRP3 is not required for dTGN formation in response to nigericin in HeLa cells. HeLa cells (without NLRP3 expression) were treated with nigericin (10 μM) for 80 min and immunostained for TGN38. Cell borders are outlined with dashed lines. b, ATP induced dTGN formation in a manner dependent on P2X7 but not NLRP3. HeLa cells or HeLa cells stably expressing P2X7–HA were incubated with ATP (5 mM) for 80 min before immunostaining for TGN38. c, Constitutively active mutants of NLRP3 can bypass the TGN-recruitment step by spontaneously forming aggregates in the cytosol. Cells stably expressing the indicated proteins were untreated or treated with nigericin (10 μM) for 80 min before immunostaining for TGN38. d, Immunoblots of cells used in Fig. 3c. e, The KKKK motif is critical for nigericin-induced NLRP3 activation. Cells stably expressing the indicated proteins were untreated or treated with nigericin (10 μM) for 60 min before being tested in the in vitro NLRP3 activation assay. f, Mutations in the KKKK motif do not compromise the ability of constitutively active NLRP3 to activate caspase-1. Cells stably expressing the indicated proteins were examined by fluorescence microscopy (top) and the in vitro NLRP3 activity assay (bottom) without stimulation. LP, NLRP3(L351P).

Extended Data Fig. 6 The polybasic region of NLRP3 functions through its positive charge.

a, Mutations of the KKKK motif to arginine do not affect nigericin-induced NLRP3 puncta formation. HeLa cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Cells with NLRP3 puncta were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). n.s., not significant (α = 0.01). 2KR, NLRP3(K127R/K128R); 3KR, NLRP3(K127R/K128R/K129R); 4KR(K127R/K128R/K129R/K130R). b, Mutations of the KKKK motif to arginine do not impair nigericin-induced NLRP3 activation. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 60 min before cell extracts were examined in the in vitro NLRP3 activity assay. c, The positive charge of the KKKK motif is important for gramicidin- and ATP-induced NLRP3 puncta formation. HeLa cells stably expressing the indicated proteins were treated with gramicidin (5 μM) (left) or ATP (5 mM) (right) for 80 min, before the percentage of cells with NLRP3 puncta was analysed as in a. d, The positive charge of the KKKK motif is essential for NLRP3 to polymerize ASC. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) or gramicidin (5 μM) for 80 min before immunostaining for both NLRP3 and ASC. The percentage of cells with NLRP3–ASC specks was analysed as in a. e, Immunoblots of cells used in Fig. 3d. f, g, The second positively charged region of NLRP3 is also important for its aggregation on dTGN and activation in a manner dependent on the number of remaining positively charged residues. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) before imaging (f) or examined by the in vitro NLRP3 activity assay (g).

Extended Data Fig. 7 NLRP3 is recruited to dTGN via binding to PtdIns4P.

a, The polybasic region of NLRP3 interacts with phospholipids in vitro through its positive charge. Left, Coomassie blue staining of purified proteins of interest, with Flag–GFP as a control. Right, PIP Strip membranes blotted with various lipids were incubated sequentially with proteins of interest, Flag antibody and HRP secondary antibody before exposure. Phospholipids with positive binding on the second PIP Strip are highlighted in red. b, Catalytically inactiveSac1 did not impair the recruitment of NLRP3 to dTGN. COS-7 cells stably expressing Flag-NLRP3 were transiently transfected with TGN38–FRB and mRFP–FKBP12–Sac1(C389S), incubated with rapamycin (1 μM) and nigericin (10 μM) for 80 min before imaging. Note that nigericin-induced dTGN in COS-7 cells sometimes looks like a single cluster because of the relatively small size of COS-7 cells and the high intensity of fluorescence signal. Under the phase contrast microscope these dTGN particles were observed to be individual vesicles adjacent to each other (data not shown). c, Only PtdIns4P phosphatase blocked NLRP3 recruitment to dTGN. Similar to b, except that indicated phosphatases were used. The target phospholipids are labelled after ‘x’. d, Immunoblots of HeLa NLRP3–GFP cells stably expressing TGN38–Sac1 or TGN38–Sac1(C389S). e, TGN-targeted Sac1 did not affect general cell morphology or nigericin-induced dTGN formation. Cells were treated with nigericin (10 μM) for 80 min before imaging with phase contrast microscopy. Cells with dTGN formation were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). n.s., not significant (α = 0.01). f, TGN-targeted Sac1 did not affect AIM2 aggregation. Cells stably expressing the indicated proteins were transfected with poly(dA:dT) (1.5 μg ml−1) for 3 h before imaging. The percentage of cells with AIM2 aggregates was analysed as in e. g, NLRP3 puncta were restricted to PtdIns4P-enriched microdomains. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Inset, higher magnification of the dTGN. Co-localization analysis was performed by calculating Pearson's correlation coefficient (threshold regression, Costes) using the Coloc 2 plugin of ImageJ. Data are presented as mean ± s.d.

Extended Data Fig. 8 Binding to PtdIns4P on dTGN is essential for NLRP3 inflammasome activation.

a, b, The KKKK motif of NLRP3 can be functionally replaced with a PtdIns4P-binding domain of OSBP (OSBP-PH). a, Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Cells with NLRP3 on dTGN were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). 2RE, NLRP3(R107/108E). Note that NLRP3(ΔKKKK OSBP-PH) that constitutively localized on intact TGN without stimulation was not counted in the quantification. b, Replacement of the KKKK motif with OSBP-PH allowed NLRP3 to be constitutively localized on TGN. Cells were treated as in a before immunostaining for TGN38. c, Mutations of OSBP-PH domain that abrogate its binding to PtdIns4P also abolish its ability to functionally rescue NLRP3(ΔKKKK). Cells were treated as in a and cell extracts were examined by the in vitro NLRP3 activity assay. d, Immunoblots for primary NLRP3-deficient BMDMs rescued with Flag–NLRP3 (wild type or mutants), which were used for experiment in Figs. 5b, 6d. The cells were infected with lentivirus encoding the indicated proteins for 6 days before immunoblotting. e, Recruitment of NLRP3 to non-PtdIns4P-enriched regions of TGN is not sufficient to support its activation. Cells stably expressing the indicated proteins were treated as in a before examination by fluorescence microscopy (left; images for NLRP3(ΔKKKK OSBP-PH) are shown in a) and the in vitro NLRP3 assay (right). f, Extracellular KCl at 30 mM was sufficient to completely block nigericin-induced K+ efflux. Cells were treated with nigericin (10 μM) for 80 min in the presence of increasing concentrations of KCl, and cell extracts were collected for measurement of intracellular K+ concentration (shown as percentage change compared to untreated sample, mean ± s.d.). Representative results from two independent experiments (each containing two samples for each condition) are shown. g, Incubation in K+-free medium induced spontaneous K+ efflux. Cells were incubated in Hanks’ buffer containing 5 mM K+ for the first two conditions, or Hanks’ buffer in which K+ was replaced by Na+ for the third condition (K+-free). The second condition also contained nigericin (10 μM). After 80 min, the cell extracts were collected for intracellular K+ measurement using methods similar to f. h, K+ efflux alone is not sufficient to activate either wild-type NLRP3 or NLRP3(ΔKKKK OSBP-PH). Cells stably expressing the indicated proteins were treated as in g before testing with the in vitro NLRP3 activity assay. i, Incubation in K+-free medium induced spontaneous K+ efflux in primary wild-type BMDMs. Cells were primed with LPS (50 ng ml−1) for 3 h, before treatment as in g for the indicated time period. Intracellular K+ concentrations were then measured and analysed as in f. j, K+ efflux alone is not sufficient to activate endogenous NLRP3 in primary wild-type BMDMs. Cells treated as in i were used for immunoblotting. Lys, lysate; sup, supernatant.

Extended Data Fig. 9 K+ efflux-independent stimuli also induced TGN dispersion and PtdIns4P-dependent NLRP3 recruitment.

a, b, K+ efflux-independent stimuli also induced NLRP3 aggregation on dTGN through the KKKK motif. HeLa cells stably expressing the indicated proteins were treated with imiquimod or CL097 (45 μg ml−1) for 80 min before imaging. High magnification images are shown in the inset. Arrows indicate representative plasma-membrane-localized NLRP3 puncta, which were separated from TGN38-positive compartments owing to the partial separation of PtdIns4P and TGN38. Cells with NLRP3 puncta on dTGN were quantified from 100 cells (n = 3, mean ± s.d.; two-sided t-test). c, Neither imiquimod- nor CL097-induced NLRP3 puncta were co-localized with mitochondria in ASC-deficient BMDMs. Cells were primed with LPS (50 ng ml−1) for 3 h and incubated with imiquimod or CL097 (45 μg ml−1) for 60 min, before immunostaining for endogenous NLRP3 and TOM20 (mitochondrial marker). d, High extracellular KCl had no effect on imiquimod- or CL097-induced NLRP3 aggregation on dTGN. HeLa cells expressing NLRP3–GFP cells were treated with imiquimod or CL097 (45 μg m−1) in the presence of KCl (0 or 30 mM) for 80 min before imaging. Results were analysed as in b. n.s., not significant (α = 0.01).

Extended Data Fig. 10 Model: NLRP3 aggregation on dTGN through PtdIns4P binding is a common cellular signal essential for the inflammasome activation by diverse stimuli.

Centre, under basal conditions, NLRP3 is diffused in the cytosol and TGN (red) remains as a single cluster attached to medial- and cis-Golgi (purple). Left, when cells are stimulated with K+ efflux-dependent stimuli such as nigericin or ATP, TGN is disassembled into multiple dispersed structures (dTGN), whereas cis- and medial-Golgi stacks remain intact. These stimuli also trigger K+ efflux, which helps recruit NLRP3 to dTGN via ionic bonding between negatively charged PtdIns4P on dTGN membranes and the positively charged polybasic region of NLRP3. Right, when cells are stimulated with K+ efflux-independent stimuli such as imiquimod and CL097, TGN is also disassembled, although more drastically. PtdIns4P is partially separated from other TGN compartments; some may be enriched on the plasma membrane. NLRP3 is then recruited to these PtdIns4P-containing microdomains through its polybasic region. For both types of stimulation, dTGN serves as a scaffold for NLRP3 to aggregate in the form of multiple puncta, which then interact with ASC to activate the downstream signalling cascade. This model shows that aggregation of NLRP3 on dTGN through PtdIns4P binding is a common cellular signal that is essential for its activation by diverse stimuli.

Supplementary information

Supplementary Information

This file contains Supplementary Results.

Reporting Summary

Supplementary Figure 1

This file contains the uncropped gel source data.

Video 1

Nigericin treatment induced the formation of large vesicles, on which NLRP3 aggregated. HeLa cells stably expressing NLRP3-GFP were examined by time-lapse live cell imaging after nigericin (10 μM) treatment. Scale bar and time lapse after nigericin treatment are shown at the bottom of the movie. Representative results from at least three independent experiments are shown.

Video 2

dTGN formation occurred earlier than NLRP3 recruitment. HeLa cells stably expressing NLRP3-GFP (green) and TGN38-mCherry (red) were examined by time-lapse live cell imaging after nigericin (10 μM) treatment. Scale bar and time lapse after nigericin treatment are shown at the bottom of the video. Representative results from at least three independent experiments are shown.

Video 3

NLRP3 puncta on dTGN vesicles didn’t colocalize with mitochondria after stimulation. HeLa cells stably expressing NLRP3-GFP (green) and MitoDsRed2 (DsRed2 protein targeted to mitochondria) (red) were stimulated with nigericin (10 μM) followed by time-lapse live cell imaging. Scale bar and time lapse after nigericin treatment are shown on the top and bottom of the video respectively. Representative results from at least three independent experiments are shown.

Video 4

dTGN formation does not require NLRP3. HeLa cells stably expressing TGN38-GFP (green) but not NLRP3 were examined by time-lapse live cell imaging after nigericin (10 μM) treatment. Differential interference contrast (DIC) channel shows that the multiple perinuclear vesicles detected in light field (e.g. Extended Data Fig. 2a) are of TGN origin. Scale bar and time lapse after nigericin treatment are shown on the top and bottom of the movie respectively. Representative results from at least three independent experiments are shown.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, J., Chen, Z.J. PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation. Nature 564, 71–76 (2018). https://doi.org/10.1038/s41586-018-0761-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-018-0761-3

Keywords

  • NLRP3 Inflammasome
  • Dt gN
  • NLRP3 Activation
  • Polybasic Region
  • Bone Marrow-derived Macrophages (BMDMs)

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing