Fig. 6: STIM1 mediates ER-mit MCS remodelling upon ER Ca²⁺ depletion.

a Representative traces of ER-mit RspA-splitFAST fluorescence in HeLa cells upon ER Ca²⁺ depletion by different treatments (arrow): 100 μM histamine (HIST); 100 μM histamine plus 100 nM thapsigargin (HIST Tg); pre-treatment (30 min) with 10 µM BAPTA-AM followed by 100 μM histamine plus 100 nM thapsigargin (BAPTA-AM); 500 μM TPEN. The box plot represents the change in ER-mit RspA-splitFAST fluorescence (ΔF/F0) 15 min after the indicated cell stimulations. b Representative confocal images of HeLa cells, expressing ER-mit RspA-splitFAST and labelled with MitoTracker, in different conditions: at basal, (CTRL); 15 min after stimulation with 100 μM histamine plus 20 μM cyclopiazonic acid (Hist CPA) and 15, 30, or 40 min after washing of Hist+CPA and re-addition of 2 mM CaCl₂ (to refill ER Ca²⁺). The box plots represent the corresponding percentages of mitochondrial surface in contact with ER. c Representative confocal images (max projection) of ER-mit RspA-splitFAST signal in pHX6743 worms, either treated with thapsigargin (Tg, see “Methods”) or not (CTRL). The box plot represents the percentage of cell area covered by ER-mit MCSs. d Representative traces of ER-mit RspA-splitFAST fluorescence upon ER Ca²⁺ depletion (Hist Tg) in HeLa cells transfected with control (siCTRL) or STIM1-, STIM2- or TMEM110-specific siRNAs. The box plot represents the change in ER-mit RspA-splitFAST fluorescence (ΔF/F0) 15 min after cell stimulation. e Traces (mean ± SEM) of either ER-mit RspA-spiltFAST or STIM1-mit RspA-splitFAST fluorescence upon Hist Tg treatment in HeLa cells. Right, representative images of HeLa cells expressing STIM1-mit RspA-splitFAST, before (t = 0 s) or 180 s after exposure to Hist Tg. f Traces (mean ± SEM) of STIM1-mit RspA-splitFAST fluorescence (F/F0) upon ER Ca²⁺ depletion (Hist Tg) in HeLa cells, co-transfected with either control or TMEM110-specific siRNA. g Representative STED images of HeLa cells untreated (NT) or stimulated for 15 min with Hist Tg, immunolabeled with αSTIM1 + αTOM20 antibodies. The box plot represents the Pearson’s co-localization coefficient between STIM1 and TOM20 signals, after 15 min in the indicated conditions (500 μM TPEN was used as an alternative treatment to deplete ER Ca²⁺). h The box plots represent mitochondrial Ca²⁺ peaks upon histamine-induced ER Ca²⁺ release in Hela cells, expressing mitochondrial Aequorin (see Methods) and transfected with control (siCTRL) or STIM1-specific siRNAs. Before the experiments, cells were transiently (15 min) treated (ERdep) or not with 500 μM TPEN. i Representative images of HeLa cells expressing STIM1-mit PRINCESS, before (t = 0 s) and 2 s after stimulation with histamine (Hist). Representative traces of OMM-PRINCESS or STIM1-mit PRINCESS fluorescence in HeLa cells, treated with histamine (Hist) in Ca²⁺-free mKRB, are shown. On the right, the box plots represent the peaks of OMM- or STIM1-mit PRINCESS fluorescence (expressed as F/F0) upon histamine stimulation (OMM-PRINCESS data as in Fig. 5f). Scale bar: 10 µm (b, c, e, g, i).