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. Author manuscript; available in PMC: 2021 Mar 18.
Published in final edited form as: Science. 2020 Sep 18;369(6510):eaas8995. doi: 10.1126/science.aas8995

Fig. 1. NLRP3 and pyrin inflammasomes, but not the AIM2 inflammasome, co-localize with the MTOC.

Fig. 1.

(A) The NLRP3, pyrin, and AIM2 inflammasome pathways triggered by nigericin or MSU, TcdB, and dsDNA, respectively. As shown below, NLRP3 and pyrin inflammasome puncta localize at the MTOC. Inflammasome activation culminates in pro-caspase-1 and pro-IL-1β processing. Upward arrows indicate processing sites. (B) Immunofluorescence images showing the co-localization of NLRP3 and ASC puncta with the centrosomal markers ninein and GTU in THP-1 cells. Blue represents nuclear staining by Hoechst 33342. (C) Line scan of intensity distribution profiles of puncta a, b, and c from (B). (D–E) Live-cell images of iBMDM-Casp-1 (D) and iBMDM-IL-1β (E) at 30 min (top panel) and 60 min (bottom panel) post-nigericin stimulation, showing co-localization of inflammasome puncta (depicted by mNeonGreen) with the MTOC (depicted by SiR-Tubulin staining that labels the microtubule network). (F) FRET analysis of caspase-1 cleavage and IL-1β processing at MTOC as a function of time for areas inside and outside the puncta in iBMDM-Casp-1 (left) and iBMDM-IL-1β (right) cells. FRET was calculated by dividing the FRET channel fluorescence intensity (donor excitation with acceptor emission) with mTurquoise2 channel fluorescence intensity (donor excitation with donor emission). Values are mean±SD for n=10–15 cells. (G–H) Recruitment of IL-1β to a region in proximity to the MTOC imaged using 3D lattice light-sheet microscopy (LLSM). iBMDM-IL-1β cells stained with SiR-Tubulin were exposed to nigericin for 12 min (G), and 23 min (H). (a–c) Representative images deconvolved using the Richardson–Lucy algorithm corresponding to a single optical plane section. The arrows highlight the MTOC and the nearby locations where IL-lβ was recruited. (d–f) Enlarged images of the regions indicated by the arrows. (I–J) Lack of co-localization of AIM2 inflammasome puncta with the MTOC in iBMDM-Casp-1 (I) and iBMDM-IL-1β (J) cells activated by dsDNA for 6 hours. (K–L) Co-localization of pyrin inflammasome puncta with the MTOC in iBMDM-Casp-1 (K) and iBMDM-IL-1β (L) cells activated by TcdB toxin for 1 hour. Images are representative of three or more independent experiments and arrowheads indicate puncta or MTOC (B, D–E, G–L). Scale bars: 10 μm (B, D–E), 5 μm (Ga–c, Ha–c, I–L), and 1 μm (Gd–f, Hd–f).