Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

Abstract

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.

Figure 1.

STIM1 accumulates near the plasma membrane before CRAC channels open. (A) Jurkat cell expressing GFP-STIM1 imaged by TIRF microscopy during whole-cell recording with a pipette solution containing 1 mM EGTA. As stores become depleted, GFP-STIM1 redistributes into puncta near the plasma membrane as shown at the indicated times. (B) CRAC currents elicited by voltage ramps from −122 to +50 mV during the image acquisitions shown in A. Data were corrected for the leak current recorded at t = 0 s and expressed relative to cell size (capacitance). (C) Time courses of I_CRAC development (measured at −122 mV) and GFP-STIM1 fluorescence (averaged across the entire cell footprint). (D) To compare the time courses of I_CRAC activation and GFP-STIM1 fluorescence, the data from C were normalized to the same minimum and maximum values. (E) Accumulation of GFP-STIM1 near the plasma membrane precedes CRAC channel activation. The difference in times for I_CRAC and GFP-STIM1 fluorescence to reach 20% of their maximum values is plotted for individual cells in response to passive depletion during whole-cell recording (left) or IP₃-induced Ca²⁺ release during perforated-patch recording (right). IP₃-induced Ca²⁺ release was triggered by cross-linking TCR with anti-CD3 mAb. The individual latencies (open circles; n = 5 cells) and mean latency (black bar) of 10 ± 2.5 s during passive depletion was comparable to the individual latencies (open squares; n = 4 cells) and mean latency (black bar) of 6.2 ± 1.8 s during IP₃-induced Ca²⁺ release. The difference in the means was not statistically significant (t test: P = 0.28). The complete image and I_CRAC sequences for A–D are shown in Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200604014/DC1). An individual cell response to TCR cross-linking is shown in Fig. S1.

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.

Figure 3.

GFP-STIM1 colocalizes with an ER marker in control and store-depleted cells. Jurkat cells coexpressing GFP-STIM1 and the ER marker DsRed2-ER were imaged with TIRF microscopy before (A–C) and after (D–F) store depletion. In cells bathed in control Ringer's solution, GFP-STIM1 (A) colocalized with the ER marker (B) as shown in the merged image (C). In cells treated with 0-Ca²⁺ Ringer's containing 1 μM TG for 5 min to deplete Ca²⁺ stores, GFP-STIM1 redistributed into puncta near the plasma membrane (D). The GFP-STIM1 puncta also overlapped with DsRed2-ER (E) as shown in the merged image (F). Similar results were seen in 13 out of 13 cells. Bars, 5 μm.

Figure 4.

Store depletion drives STIM1 puncta formation without bulk ER movement toward the plasma membrane. TIRF images of a Jurkat cell coexpressing GFP-STIM1 and DsRed2-ER before and after store depletion. The images shown were collected at the times indicated by the arrows in C. Treatment with 1 μM TG in 0-Ca²⁺ Ringer's solution depleted Ca²⁺ stores and caused GFP-STIM1 to redistribute from a diffuse ER localization into bright puncta near the plasma membrane (A) without a significant increase in DsRed2-ER fluorescence (B). (C) After store depletion, GFP-STIM1 fluorescence (closed squares) increased without a corresponding increase in DsRed2-ER fluorescence (open circles) in the same cells (n = 14). Bar, 5 μm.

Figure 5.

Ultrastructural localization of ER in Jurkat cells before and after store depletion. Jurkat cells were transiently transfected with HRP-ER plasmid and processed for HRP cytochemistry and EM. (A and B) In resting cells with full Ca²⁺ stores, HRP-ER was located in the nuclear envelope and numerous cytoplasmic tubules (arrowheads), as well as in tubules closely associated with the plasma membrane (arrows). Large, round electron-dense structures (asterisks) were present in HRP-expressing cells as well as in untransfected cells, indicating that they are probably DAB-reactive peroxisomes or lysosomes. (C and D) In cells treated with 1 μM TG for 15 min to deplete stores, the overall distribution of HRP-ER was similar to that of resting cells, with dense reaction product present in both cytosolic and plasma membrane–associated tubules. The area outlined by the box in C is shown at higher magnification in D. n, nucleus; pm, plasma membrane. Bars: (A and C) 2 μm; (B and D) 200 nm.

Figure 6.

Ultrastructural localization of HRP-STIM1 in control and store-depleted Jurkat cells. (A–C) In resting cells with full Ca²⁺ stores, HRP-STIM1 was localized in tubules throughout the cytoplasm, having a similar distribution as tubules marked by HRP-ER (Fig. 5, A and B). (D–H) In cells treated with 1 μM TG for 15 min to deplete stores, HRP-STIM1 was concentrated in tubules closely associated with the plasma membrane (arrows), whereas weak, diffuse staining was observed in cytoplasmic ER tubules (arrowheads). m, mitochondrion; n, nucleus; pm, plasma membrane. Bars: (A, D, and E) 2 μm; (A [inset], B, C, F, and G) 200 nm; (H) 100 nm.

Figure 7.

Store depletion enhances junctional contacts between the ER and the plasma membrane. Quantitative analysis of EM data from experiments like those in Figs. 5 and 6. (A) For each section where a nucleus was clearly visible, the lengths (red dashed lines) of all HRP-containing tubules located within 50 nm of the plasma membrane (green dotted line) were measured. (B) The total length of junctional HRP-tubules per section (normalized to the plasma membrane circumference) was averaged in HRP-ER–expressing cells with full (black; n = 17 cell sections) or depleted stores (blue; n = 15) and in HRP-STIM1–expressing cells with full (green; n = 16) or depleted stores (red; n = 21). The total length of junctional contacts increased after store depletion (t test: *, P = 0.02; **, P = 0.007). HRP-STIM1 overexpression did not significantly change the amount of junctional ER in resting cells (black vs. green). (C) Store depletion also increased the number of junctional contact sites (as measured by the number per micrometer of plasma membrane; t test: *, P = 0.006; **, P = 0.02). (D) Store depletion significantly increased the mean junctional tubule length in HRP-STIM1–expressing cells (ANOVA: **, P = 10⁻²⁹; gray, n = 183 tubules; blue, n = 228; green, n = 133; red, n = 288; a total of 832 tubule lengths from 69 micrographs of cell sections were measured). (E) The distributions of the lengths of junctional HRP-ER tubules in resting cells (gray) and store-depleted cells (blue) were similar. (F) The lengths of junctional HRP-STIM1 tubules increased after store depletion (green, control; red, store depleted). All error bars show SEM. PM, plasma membrane.