Fig. 6: Activated Ezrin inhibits SOCE by directly binding to Orai1 and promotes retraction of membrane blebs.

a Representative still images of membrane blebbing in DLD1 cells expressing Lifeact-RFP and GFP-E-Syt1 from five independent experiments. See also Supplementary Movie 6. b, c DLD1 cells expressing Lifeact-RFP with either GFP-E-Syt1 (b) or GCaMP6s (c) were treated with the actin polymerization inhibitor Latrunculin B (LatB, 10 µM). Arrowheads show disrupted actin cortex. Results shown are representative of three independent experiments. See also Supplementary Movie 7. d–f Biochemical analyses of ezrin’s role in STIM1-Orai1 complex formation. d Orai1-HA was co-expressed with either GFP or GFP-ezrin. Inputs and HA immunoprecipitates were immunoblotted with antibodies against HA and GFP. e STIM1-FLAG and Orai1-HA were co-expressed with either GFP or GFP-ezrin. Inputs and FLAG immunoprecipitates were immunoblotted with antibodies against FLAG, HA, and GFP. f GFP expression levels (shaded in green) and co-precipitated Orai1-HA (individual measurements) were quantified and normalized to GFP-expressing control. Data presented are means ± SD based on the values from four independent experiments. g, h Proximity ligation assay (PLA) for in situ detection of Orai1-STIM1 interaction. Representative images from five independent experiments are shown in g. h Quantification of PLA signals in expanding (low Lifeact intensity) and retracting (high Lifeact intensity) blebs based on data shown in g and Supplementary Fig. 6a. STIMATE-STIM1 and Claudin-3-STIM1 are positive and negative controls, respectively. N = 25 independent blebs. Individual data points are plotted with the means ± SD. i Membrane blebbing in Ezrin KO cells expressing GFP- PLCδ-PH and Sec61β-mCherry (upper panels) and in WT DLD1 cells expressing constitutive active Rnd3 (S240A) and Sec61β-mCherry (lower panels). Arrowheads indicate persistent ER-PM contact sites. Results shown are representative of three independent experiments. j Representative images from three independent experiments of DLD1 cells expressing constitutive active Ezrin (T567E) and Sec61β-mCherry. a, g (middle and bottom panels), i, j (three right panels) Scale bar, 2 μm. b, c, g (top panels), and j (left panel) Scale bar, 10 μm. f, h ****P < 0.0001 (Two-sided, unpaired Student’s t test). Times shown are relative to drug treatment (b, c) or the first image (i). Source data are provided as a Source Data file.