Fig 1: Rab11a mediates endosomal TWIK2 plasmalemma translocation and NLRP3 inflammasome activation on ATP challenge of macrophages.(A) Confocal images of Rab11a immunostaining in mouse monocyte-derived macrophages (MDMs) from three mice before and after ATP challenge. Rab11a (green) distribution was identified with fluorescent immunostaining with anti-Rab11a antibody (ab65200 from Abcam). Red arrows show translocated Rab11a after ATP challenge. Scale bar = 10 µm. We observed dispersed distribution and plasmalemma translocation of Rab11a after ATP challenge (bottom panel). (B) Summary of Rab11a plasmalemma translocation as shown in (A). ***p < 0.001 compared with control group. (C–F) Rab11a-, P2X7-, and Ca2+-dependent TWIK2 plasma membrane translocation induced by ATP. (C) Confocal images of TWIK2 plasmalemma translocation on ATP challenge in mouse RAW 264.7 macrophage cells transfected with TWIK2-GFP plasmids (green) under different conditions as indicated. Cells pretreated with siRab11a (or scRNA as control) or siP2X7 (or scRNA as control) for 48 hr or pretreated with BAPTA-AM (or phosphate-buffered saline [PBS] as control) for 20 m were stimulated with ATP or PBS (control) for 15 m; then the cells were imaged using a confocal microscope. Red arrows showing the translocated TWIK2 after ATP challenge. Scale bar = 5 µm. (D–F) Quantification of the TWIK2 plasmalemma translocation under different conditions (pretreated with siRab11a (D), pretreated with siP2X7 (E), and pretreated with BAPTA-AM (F)) based on the confocal images as shown in (E). ***p < 0.001. Depletion of Rab11a or P2x7 or blocking intracellular Ca2+ increase (by BAPTA-AM) significantly reduced TWIK2 plasmalemma translocation after ATP challenge. (G, H) Reduced ATP-induced K+ outward current in RAW 264 macrophages treated with dominant-negative Ra11a (Rab11a DN) for 48 hr. Whole-cell current was recorded with patch clamp as described in Figure 3B. (G) Representative I–V plot of whole-cell current. (H) Summary from experiments displayed in (G). ***p < 0.001 compared with WT basal, n = 5. ###p < 0.001 compared with WT ATP group, n = 5. Cells pretreated with Rab11a DN showed significantly decreased current induced by ATP. (I–O) Rab11a-dependent NLRP3 inflammasome activation induced by ATP in macrophages. These experiments were carried out in MDMs treated with siRNA targeting mouse Rab11a (siRab11a). (I) Representative western blot results from three independent experiments with MDMs from three mice showing reduced caspase 1 activation (reduced Casp-1 p20), IL-1ß maturation (reduced IL-1ß p17), and depletion of Rab11a in cells treated with siRab11a in MDMs whereas NLRP3 expression was not affected by siRab11a. MDMs pretreated with siRab11a for 48 hr were primed with lipopolysaccharide (LPS; 3 hr) and subsequently challenged with ATP (5 mM) for 30 m. Cell lysates were immunoblotted with indicated antibodies (anti-TWIK2 or anti-IL1ß or anti-Rab11a or anti NLRP3). (J–M). Quantification of results shown in (I). *p < 0.05, **p < 0.01, ***p < 0.001, n = 3. The reductions in Casp-1 p20, IL-1ß p17, Rab11a, but not NLRP3 expression were seen in cells treated with siRab11a. Reduced IL-1ß (N) and IL-18 (O) release in cells treated with siRab11a. MDMs pretreated with siRab11a for 48 hr were primed with LPS (3 hr) and subsequently challenged with ATP (5 mM) for 30 m. Release in IL-1ß and IL-18 in the supernatant was measured with ELISA. *p < 0.05, **p < 0.01, n = 3. See also Figure 5—figure supplement 1. Figure 5—source data 1.Rab11a mediates endosomal TWIK2 plasmalemma translocation and NLRP3 inflammasome activation on ATP challenge in macrophages.Related to Figure 5I. Rab11a-dependent NLRP3 inflammasome activation induced by ATP in macrophages. Inhibited NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) treated with siRNA targeting mouse Rab11a (siRab11a). Representative results of western blot from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20) and IL-1ß maturation (reduced IL-1ß p17) and Rab11a knocking down after cells were treated with siRab11a in MDMs, but the NLRP3 expression was not affected by siRab11a treatment. MDMs pretreated with siRab11a for 48 hr were primed with lipopolysaccharide (LPS; 3 hr) and subsequently challenged with ATP (5 mM) for 30 min. Cell lysates were immunoblotted with indicated antibodies (anti-TWIK2 or anti-IL1ß or anti-Rab11a or anti NLRP3).
Fig 2: Endosomal localization of intracellular TWIK2 determined with fluorescent immunostaining of macrophages.TWIK2 intracellular localization was determined with fluorescent immunostaining with TWIK2 antibody along with other various antibodies against some specific vesicular proteins and imaged with confocal microscope. RAW 264.7 macrophages transfected with TWIK2 plasmids were fixed/permeabilized followed by immunostaining. TWIK2 (green) was identified with anti-TWIK2 antibody (LSBio #LS-C110195-100) in (A–D). Early endosomes (EE, red) were identified with antibody (clone1D4B) against EEA1 (C45B10 from Cell Signaling Technology) in (A). Recycling endosomes (RE, red) were identified with antibody against Rab11a (ab65200 from Abcam) in (B). Lysosomes (red) were identified with antibody against lysosomal membrane protein LAMP1 (D2D11 from Cell Signaling Technology) in (C). ER was identified with antibody against Protein Disulfide Isomerase (PDI; C81H6 from Cell Signaling Technology) in (D). Scale bar = 10 µm. (E) Quantification of co-localization of TWIK2 with cell organelles based on the confocal images as shown in (A–D). ***p < 0.001. The localization of TWIK2 with organelles (green) was seen in both the EE and RE (white arrows) but much less in lysosomes or ER.
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