Fig. 1.

ER Ca²⁺ controls STIM1 oligomerization and puncta formation. (A) Schematic representation of a FRET-based assay for STIM1 oligomerization. (B) STIM1 oligomerization and puncta formation in YFP-STIM1 and CFP-STIM1 cotransfected RBL cells. Confocal images were acquired near the adhesion surface before and 90 s after antigen (2 μg/ml dinitrophenol-BSA) stimulation. (Scale bar, 10 μm.) (C) The average STIM1 FRET_E trace of 31 RBL cells. The average and SE of t_1/2 are shown. (D) Confocal images of YFP-STIM1 and CFP-ER marker cotransfected RBL cells were acquired near the adhesion surface before and 90 s after antigen stimulation. The overlay images show the colocalization of STIM1 (green) and the ER marker (red). (Scale bar, 5 μm.) (E) YFP-STIM1 and CFP-STIM1 cotransfected HeLa cells were stimulated with 100 μM histamine (Hist.) plus 5 μM BHQ in a Ca²⁺-free buffer for 4 min. Cells were then washed three times, and 10 mM Ca²⁺ was added back. Confocal images were acquired near the adhesion surface before (Basal), 75 s after stimulation (Store depleted), and 70 s after Ca²⁺ readdition (Store refilled). (Scale bar, 10 μm.) (F) The average STIM1 FRET_E trace of 10 HeLa cells. The average and SE of t_1/2 are shown.