Fig. 7.

Depletion of ER Ca²⁺ stores causes native STIM1 to accumulate at functional ER-PM junctions adjacent to immobile IP₃R puncta. a, b Representative TIRFM images of EGFP-IP₃R1 HeLa cells fixed and immunostained for STIM1 before (a) or after treatment with thapsigargin (Tg, 1 µM, 15 min) to deplete the ER of Ca²⁺ (b). Overlaid images of Tg-treated cells show no significant co-localization of STIM1 and IP₃R puncta (Pearson’s coefficient with Costes’ automatic threshold: 0.331 ± 0.026, n = 7 cells). c Distribution of mobile (green and magenta) and immobile (white) IP₃R puncta in Tg-treated cell. Scale bars (a–c) = 10 µm. d Enlargements of the boxed regions in b show that immobile IP₃R puncta (identified before fixation (c), with all shown by arrowheads) abut STIM1 puncta without coinciding with them. Scale bars = 2 µm. e Fluorescence intensity profiles for EGFP-IP₃R1 and STIM1 across the lines shown in d. Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP₃R are shown. f Co-localization of CFP-STIM1 (pseudocoloured green) and mCherry-Orai1 (red) puncta in a Tg-treated HeLa cell. We used tagged proteins because available antibodies do not reliably detect endogenous Orai1. Scale bar = 10 µm (2 μm in enlargement)