Figure 2.

GFP-STIM1 puncta in store-depleted cells are intracellular. Three Jurkat cells expressing GPI-GFP, GFP-STIM1, or cytosolic GFP were imaged at 20-s intervals by TIRF microscopy. The images in A–C were collected at the time points indicated by the arrows in D. (A) Cells were pretreated for 10–15 min with 0-Ca²⁺ Ringer's solution and 1 μM TG, pH 7.3, to deplete Ca²⁺ stores and redistribute GFP-STIM1 into puncta. (B) Perfusion with pH 5.2 solution immediately quenches the GPI-GFP fluorescence without changing GFP-STIM1 and cytosolic GFP fluorescence. (C) GFP-STIM1 and cytosolic GFP are quenched by the pH 5.2 solution only in the presence of nigericin and monensin, which permeabilize the cells and equilibrate the intracellular and extracellular pH. (D) The time course of mean fluorescence changes for cells expressing GPI-GFP (open circles; n = 7), GFP-STIM1 (closed circles; n = 5), or cytosolic GFP (open triangles; n = 3). Values are normalized to fluorescence at t = 100 s. The complete image sequence for the experiment in A–C is shown in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200604014/DC1). Bar, 5 μm.