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Figure S2. Fluorescence-quenching measurements using wide-field epifluorescence microscopy to assay for plasma membrane-localized GFP-STIM1. Three Jurkat cells expressing GPI-GFP, GFP-STIM1, or a cytosolic GFP were imaged at 20-s intervals using wide-field epifluorescence microscopy. In each example, the plane of focus passed through the middle of the cell. The images in A-D were collected at the time points indicated by the arrows in E. (A) In resting cells with full Ca²⁺ stores, GFP-STIM1 was distributed throughout the ER, whereas cytosolic GFP was restricted to the cytoplasm and GPI-GFP was located almost exclusively on the plasma membrane. (B) Perfusion with 0-Ca²⁺ Ringer’s containing 1 µM TG, pH 7.3, depleted intracellular Ca²⁺ stores and caused GFP-STIM1 to form bright puncta located near the plasma membrane (arrowheads), whereas the distributions of GPI-GFP and cytosolic GFP were unchanged. (C) Perfusion with pH 5.2 solution quenched the fluorescence of the extracellular-facing GPI-GFP within one frame but did not immediately reduce the fluorescence of GFP-STIM1 or cytosolic GFP. (D) GFP-STIM1 and cytosolic GFP were quenched after the addition of nigericin and monensin to equilibrate intracellular pH with the extracellular pH 5.3 solution, indicating an intracellular location for these proteins. (E) Time course of mean fluorescence changes in cells expressing GPI-GFP (blue; n = 7), GFP-STIM1 (green; n = 5), or cytosolic GFP (black; n = 7). Values are normalized to fluorescence at t = 540 s. Bar, 5 µm. The complete image sequence for the experiment in A-D is shown in Video 4.