Research ArticleCalcium signaling

ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation

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Science Signaling  18 Dec 2018:
Vol. 11, Issue 561, eaat0358
DOI: 10.1126/scisignal.aat0358

A snapshot after T cell activation

Stimulation of the T cell receptor (TCR) initially elicits local Ca2+ signals at the plasma membrane that propagate to trigger global Ca2+ signals that fully activate T cells. Using high-resolution imaging and biochemical techniques to analyze mouse T cells and a human T cell line, Diercks et al. identified the proteins that generate Ca2+ signals within the first second after T cell activation. In unstimulated cells, the plasma membrane–localized Ca2+ channel ORAI1 was constitutively associated with the ER-localized Ca2+ sensors STIM1 and STIM2 and generated brief, spontaneous, and small-amplitude Ca2+ signals. Stimulation of the TCR induced production of the second messenger NAADP, which promoted Ca2+ release through the ryanodine receptor RYR1, ensuring the Ca2+ influx required for the sustained T cell activation. These results identify the Ca2+-handling proteins and show how they are poised to respond to TCR stimulation.

Abstract

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

INTRODUCTION

In nonexcitable cells such as T cells, sustained Ca2+ signaling is mediated by store-operated Ca2+ entry (SOCE) that consists of two phases. Upon T cell receptor (TCR) stimulation, the second messengers nicotinic acid adenine dinucleotide phosphate (NAADP), d-myo-inositol 1,4,5-trisphosphate (IP3), and cyclic adenosine 5′-diphosphate–ribose (cADPR) are formed sequentially and bind to their respective target Ca2+ channels, such as ryanodine receptors (RYRs) and IP3 receptors (IP3Rs), located in the endoplasmic reticulum (ER) (13). These Ca2+ channels open, and ER-derived Ca2+ signals in the cytoplasm occur. The concurrent decrease of Ca2+ concentrations in the ER is sensed by stromal interaction molecule 1 (STIM1) and STIM2, which undergo conformational changes and activate ORAI1 and ORAI2 channels in the plasma membrane (PM), resulting in SOCE (47).

T cell activation is initiated by cell-cell contact with an antigen-presenting cell. Then, ligation of the TCR/CD3 complex by major histocompatibility complex molecules evokes juxtacrine signaling. The first intracellular signals occurring within tens and hundreds of milliseconds are local Ca2+ release events, termed Ca2+ microdomains [(8), reviewed in (9)]. Using a high-resolution live cell–imaging technique, we have shown that RYR1 and NAADP are important players in Ca2+ microdomains, early during T cell activation (8). In addition to RYR1, Ca2+ entry also contributes to Ca2+ microdomain formation, as shown in T cells in nominally Ca2+-free extracellular solution (8).

In this study, we sought to identify the Ca2+ entry component involved in the subsecond time window after TCR stimulation. Further, we aimed to elucidate the mechanism underlying transition from unstimulated to stimulated T cells with respect to Ca2+ signaling.

RESULTS

Using high-resolution Ca2+ imaging (8) in unstimulated wild-type (WT) T cells, infrequent small Ca2+ microdomains spontaneously occurred close to the PM (Fig. 1A, top, arrowheads). These spontaneous Ca2+ microdomains were characterized by an amplitude of 290 ± 12 nM and a frequency of about four signals per second within the confocal plane of the cell (Fig. 1B). In contrast, primary Orai1−/− and Stim1−/−/Stim2−/− T cells displayed significantly decreased numbers of spontaneous Ca2+ microdomains (Fig. 1, A and B). The amplitude of Ca2+ microdomains was only affected in Stim1−/−/Stim2−/− T cells, but not in Orai1−/−, Stim1−/−/Stim2−/−, or Ryr1−/− T cells (Fig. 1C). Moreover, blockade of NAADP-evoked Ca2+ release by the specific antagonist BZ194 [3-carboxy-1-(2-(octylamino)-2-oxoethyl)pyridin-1-ium bromide] (10) did not alter spontaneous Ca2+ microdomains, indicating that the NAADP/RYR1 signaling axis is not involved in spontaneous Ca2+ microdomains.

Fig. 1 Spontaneous Ca

2+ microdomains in unstimulated T cells are dependent on expression of ORAI1 and STIM1/2. (A) Ca2+ microdomains in an unstimulated WT T cell (top), Orai1−/− T cell (middle), and Ryr1−/− T cell (bottom). Arrowheads indicate Ca2+ microdomains directly at the PM. (B) Characteristics of Ca2+ microdomains in primary murine WT (n = 69), Orai1−/− (n = 28), Stim1−/− (n = 24), Stim2−/− (n = 39), and Stim1−/−/Stim2−/− (n = 46) T cells in a 5-s time period. Comparison of the number of signals per confocal plane and frame (data represent means ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test. (C) Mean [Ca2+]i of the cells used in (B). **P < 0.01, ***P < 0.001, Kruskal-Wallis test. (D to G) Kinetic analysis of the 5-s time period. (D and F) Time course of spontaneous Ca2+ signals in a confocal sublayer (as indicated). (E and G) Statistical analysis was performed by comparing WT against all other genotypes (as indicated) with Mann-Whitney U test across all time points. **P < 0.005, ***P < 0.001. ns, not significant.

Further, subsecond kinetics of spontaneous Ca2+ microdomain formation was analyzed in the subplasmalemmal space. The analyzed sublayer was ~423 ± 32 nm in depth and comprised an area spanning a 90° angle of the confocal plane analyzed (Fig. 1, D to G, insets; for analysis of T cells using dartboard segments, see also fig. S1). In this layer, almost no Ca2+ microdomains were present in Orai1 and Stim1/Stim2-deficient T cells, which differ significantly from WT T cells (Fig. 1F). In contrast, Ryr1 deletion did not significantly affect spontaneous subplasmalemmal Ca2+ microdomains (Fig. 1, A, bottom, and D). These results again suggest that basal Ca2+ entry microdomains are driven by preactivated ORAI1 and STIM1/STIM2 clusters of unstimulated T cells, whereas RYR1 or NAADP does not appear to be critical to this process.

As an independent approach, spontaneous Ca2+ microdomains were analyzed in Jurkat T cells stably transfected with ORAI1 fused to a genetically encoded Ca2+ indicator for optical imaging (GECO), which detects Ca2+ entry through ORAI1 channels (11). Spontaneous local and oscillatory Ca2+ entry events through ORAI1 occurred in the presence of 1 mM extracellular Ca2+ (Fig. 2, A and B, first row). Chelating free extracellular Ca2+ with EGTA markedly decreased these spontaneous Ca2+ entry events (Fig. 2, A and B, second row). Inhibition of Ca2+ entry using the specific Ca2+–release activated Ca2+ (CRAC) channel inhibitor Synta66 (Fig. 2, A and B, third row) further confirmed the involvement of ORAI1. Rapid chelation of free cytosolic Ca2+ by preloading the cells with 5 μM BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] also largely diminished ORAI1-GECO signals, indicating that the freely diffusible BAPTA-free acid competes effectively with the GECO construct for free Ca2+ ions entering the cell through ORAI1 (Fig. 2, A and B, fourth row). Inhibition of NAADP signaling by BZ194 (10) did not affect spontaneous Ca2+ microdomains (Fig. 2, A and B, bottom row). Quantification of spontaneous local Ca2+ entry events at four different regions of interest (ROIs) revealed significant decreases in the absence of extracellular Ca2+, buffering of free cytosolic Ca2+ with BAPTA or upon inhibition by Synta66 (Fig. 2C), confirming the Ca2+ microdomain data in Fig. 1 and further suggesting that preformed clusters of ORAI1 and STIM1 are constitutively active at single sites in unstimulated T cells.

Fig. 2 Spontaneous local Ca

2+ entry events mediated by ORAI1. (A) Nonstimulated Jurkat G-GECO1.2-Orai1 T cells were imaged in 1 mM Ca2+ buffer (control), in nominal Ca2+-free buffer with 1 mM EGTA (no [Ca2+]ex), or in 1 mM Ca2+ buffer containing 100 μM Synta66. Jurkat G-GECO1.2-Orai1 T cells were loaded with 5 μM BAPTA-AM or incubated with 500 μM BZ194 overnight. The time points indicated in (A) are represented in (B) by dashed lines. (B) F/F0 tracings of ROIs (5 × 5 pixel) from the five representative Jurkat G-GECO1.2-Orai1 T cells in (A). Arrowheads in (A) indicate the ROIs used in (B). Noise in the tracing was reduced by a moving average filter (width = 5). (C) Statistical analysis of four ROIs per cell (20 × 10 pixel) over 300 s from control (n = 31), no [Ca2+]ex (n = 17), Synta66 (n = 18), BAPTA (n = 18), and BZ194 (n = 8) cells. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test, statistically significant differences between the number of signals per ROI per second. Scale bars, 10 μm (A).

We next investigated preformed clustering of STIM1 and ORAI1 in unstimulated T cells. We used (i) immunoprecipitation, (ii) Förster resonance energy transfer (FRET), or (iii) stimulated emission-depletion (STED) super-resolution confocal microscopy. First, we performed immunoprecipitation assays with beads coupled with either ORAI1 or STIM1 antibodies and fragmented membranes obtained from Jurkat T cells (Fig. 3, A to C). In assays using cells stimulated with thapsigargin (Tg) for 10 min, ORAI1 was detected in STIM1 pull-downs, and STIM1 was detected in ORAI1 pull-downs (Fig. 3B). In assays using unstimulated control samples, much weaker but detectable signals were obtained, suggesting preformed STIM1/ORAI1 clusters in unstimulated Jurkat T cells (Fig. 3B). Similarly, in unstimulated primary WT T cells, Orai1 was detected in STIM1 pull-downs (Fig. 3, C and D). We next used FRET imaging to further verify the formation of functional STIM1-ORAI1 complexes. FRET signals were detected in unstimulated Jurkat T cells at the PM (Fig. 3, E, top, and F). These signals were strongly increased upon stimulation with Tg for 10 min (Fig. 3, E, bottom, and F). STED super-resolution confocal microscopy with 40-nm spatial resolution in the xy dimension (fig. S2, A to D) revealed PM/sub-PM areas (Fig. 3, G and H) either with strong colocalization of STIM1 and ORAI1 (Fig. 3G, “merge,” region 1) or with ORAI1 alone (Fig. 3G, merge, region 2). Although those areas without colocalization of ORAI1 and STIM1 are unlikely to play major roles for the spontaneous Ca2+ microdomains, patches of the PM of unstimulated primary T cells, where STIM1 and ORAI1 are strongly colocalized, are very likely the sites of such events. These patches had a length of ~1 μm and penetrated ~300 nm into the cytoplasm (Fig. 3I). Within these patches, STIM1 and ORAI1 clusters were adjacent and partially overlapped, with a high colocalization coefficient of 0.61 ± 0.03 (Fig. 3, G to J). These results substantiate the notion of a functional interaction between STIM1 and ORAI1 in spontaneous Ca2+ microdomains at ER-PM junctions.

Fig. 3 ORAI1 and STIM1 form functional clusters in unstimulated T cells.

(A to D) Immunoprecipitation (IP) experiments in Jurkat T cells (A and B) and primary murine WT T cells; representative experiments from at least three experiments in Jurkat T cells and primary T cells (C and D). (A) Detection of STIM1 and ORAI1 in membranes of Jurkat T cells. (B) Representative pull-down of fragmented membranes obtained from unstimulated Jurkat T cells using beads coupled with either anti-ORAI1 (top) or anti-STIM1 (bottom) (−Tg). (C) Detection of STIM1 and ORAI1 in whole-cell lysates from primary murine WT T cells. (D) Pull-down of fragmented membranes obtained from unstimulated primary WT T cells using beads coupled with anti-STIM1 (bottom) (−Tg). The interaction of ORAI1 with STIM1 was assessed in Jurkat and primary murine T cells stimulated with Tg for 10 min (+Tg) as a positive control. (E) Representative Jurkat T cells coexpressing STIM1 CFP (cyan fluorescent protein) and ORAI1 YFP (yellow fluorescent protein). Basal STIM CFP, ORAI1 YFP, and FRET signal in unstimulated cells (top) and after 10-min of stimulation by Tg (bottom). Scale bars, 5 μm. (F) Statistical analysis of FRET at the PM after correction for spectral cross-talk (bleedthrough) from unstimulated (basal n = 20) and Tg-stimulated Jurkat cells (n = 18). Background corresponds to nonspecific FRET within the cytosol of both groups. Data represent means ± SEM. *P < 0.05, Kruskal-Wallis test. (G) Colocalization of STIM1 (green) and ORAI1 (red) in a representative primary WT T cell, showing single optical channels, merge of both optical channels, and colocalization (white). Scale bar, 2 μm. (H) Magnification of a high (1) and a low (2) colocalization area of the ROIs depicted in (G). Scale bars, 100 nm. (I) STIM1-ORAI1 plot profiles of the arrow shown in image 1 in (H). (J) Statistical colocalization analysis of STIM1-ORAI1 (n = 17 cells). Data represent means ± SEM. ****P < 0.0001, unpaired t test.

Stimulation of WT T cells using microbeads coated with CD3 and CD28 antibodies resulted in an immediate increase in the number and amplitude of small Ca2+ microdomains within the first 15 s of stimulation, whereas at about 25 s global Ca2+ signaling began (Fig. 4A, top, and movie S1). Analysis of ROIs set below the PM, in the cytosol, or in the nucleus indicated that [Ca2+]i rapidly increased below the PM at site of stimulation and then nearby in the cytosol, whereas at the opposite side of the cell below the PM or in the nucleus, Ca2+ microdomains were not detected within 15 s (Fig. 4B). In contrast to WT T cells, almost no Ca2+ microdomains were detected in Orai1−/− T cells (Fig. 4, A, bottom, and C, and movie S2). In the few cases where Ca2+ microdomains were recorded in Orai1−/− T cells, they preferentially occurred more deeply within the cytosol (fig. S3, A and B). The numbers of Ca2+ microdomains that developed within 15 s of TCR/CD28 stimulation were significantly decreased not only in Orai1−/− T cells but also in Stim2−/− and Stim1−/−/Stim2−/− T cells, but not in Stim1−/− T cells (Fig. 5A and movie S3). Further, Orai1−/− or Stim1−/−/Stim2−/− T cells also showed a significant decrease in the amplitude of the microdomains (Fig. 5B). Likewise, the percentage of Stim1−/−/Stim2−/− T cells that responded with one or more microdomains within 15 s after stimulation was about fourfold lower compared to WT T cells (Fig. 5C). Representative examples for Stim1−/−, Stim2−/−, or Stim1−/−/Stim2−/− T cells demonstrate the largely diminished number and amplitude of Ca2+ microdomains (fig. S4, A to F). This effect was also seen when cells were analyzed in a “dartboard” projection fashion (fig. S5), in which the confocal plane analyzed in each cell was split into segments of identical area, each displaying the mean number of local Ca2+ signals in a color-coded fashion (fig. S5). No difference in the number of Ca2+ microdomains was observed in T cells upon deletion of Orai2, although ORAI2 synergizes with ORAI1 to mediate SOCE after store depletion (fig. S6) (7).

Fig. 4 ORAI1 is critical for initial Ca

2+ microdomain formation upon TCR/CD28 stimulation. (A) Initial Ca2+ microdomains in a WT T cell (top) and an Orai1−/− T cell (bottom) upon anti-CD3/anti-CD28 stimulation (bead contact indicated in the scheme) and a magnified region near the bead contact site. (B and C) Respective Ca2+ tracings of four ROIs (3 × 3 pixel) shown in (A) (top, WT; bottom, Orai1−/−). Dashed line indicates time point of bead contact.

Fig. 5 Effect of Stim1

−/−, Stim2−/−, and Stim1−/-Stim2−/− on formation of Ca2+ microdomains after TCR/CD28 stimulation. (A to C) Characteristics of initial Ca2+ microdomains in primary murine WT (n = 69), Orai1−/− (n = 28), Stim1−/− (n = 24), Stim2−/− (n = 39), and Stim1−/−/Stim2−/− (n = 46) T cells within 15 s after bead contact. Comparison of the number of signals per confocal plane and frame (A), the Ca2+ amplitude (B), and the percentage of T cell that responded with one or more microdomains within 15 s after stimulation (C) (data represent means ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test. (D) Primary WT, Orai1−/−, Stim1−/−, Stim2−/−, and Stim1−/−/Stim2−/− T cells were stimulated by anti-CD3/anti-CD28–coated beads. Subcellular layers at the PM (as indicated) were analyzed in the first second before and after bead contact. (E) Statistical analysis was performed comparing WT with all other genotypes (as indicated) with Mann-Whitney U test across all time points after activation. *P < 0.05, **P < 0.005, ***P < 0.001. (F) Ca2+ microdomain numbers were compared either before stimulation (−5 to 0 s) or in 5-s intervals after stimulation, as indicated. Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test, statistically significant differences between the number of signals per confocal plane per frame.

How do Ca2+ microdomains occur and spatiotemporally propagate within activated T cells? To address this question, subsecond kinetics of Ca2+ microdomain formation after T cell activation were analyzed below the PM of T cells close to the site of TCR/CD28 stimulation. The numbers of Ca2+ microdomains (calculated per confocal sublayer and frame) increased ~100 ms after TCR/CD28 stimulation in WT T cells (Fig. 5, D and E), and these numbers were significantly higher than in Orai1−/−, Stim1−/−, Stim2−/−, or Stim1−/−/Stim2−/− T cells (Fig. 5E). To elucidate potentially differential role(s) of STIM1 and STIM2 in this initial phase of T cell activation, Ca2+ microdomain formation was analyzed in temporal segments, either before stimulation or in 5-s segments after stimulation (Fig. 5F). Before stimulation and within the first 5 s after stimulation, Stim1−/−, Stim2−/−, or Stim1−/−/Stim2−/− T cells showed markedly decreased numbers of Ca2+ microdomains (Fig. 5F). This effect was stronger and statistically significant for Stim2−/− and Stim1−/−/Stim2−/− T cells within 5 s after stimulation. Within the next four 5-s segments, the extent of the difference in the number of Ca2+ microdomain numbers became less severe in Stim1−/− T cells, whereas Stim1−/−/Stim2−/− T cells had the same largely and significantly reduced numbers of Ca2+ microdomains (Fig. 5F), suggesting that, over time, STIM2 could functionally replace STIM1. Between 55 and 60 s in either Stim1−/− or Stim2−/− T cells, Ca2+ microdomain formation was similar to WT cells (Fig. 5F), suggesting that, at this later time point, each STIM protein may replace each other, whereas the phenotype of the Stim1−/−/Stim2−/− double knockout was similarly severe for all temporal segments (Fig. 5F).

We next asked how Ca2+ release and entry systems cooperate to form Ca2+ microdomains. In previous work, we have suggested that RYR1, possibly activated by NAADP, is the main Ca2+ release channel involved in Ca2+ microdomain formation upon TCR/CD28 stimulation (8). Here, we confirmed this finding by showing that the percentage of responding cells, as well as the number of microdomains per cell in the first 15 s of T cell activation, was decreased in Ryr1−/− T cells (Fig. 6A). Analysis of subsecond kinetics of [Ca2+]i also revealed significantly fewer Ca2+ microdomains in the first second after TCR/CD28 stimulation compared to WT T cells (Fig. 6B and fig. S7A). Similarly, blocking NAADP signaling using the NAADP antagonist BZ194 (10) resulted in a significantly decreased percentage of responding cells and decreased number of Ca2+ microdomains in the first 15 s of T cell activation (Fig. 6C). Subsecond kinetics of Ca2+ microdomain formation provides further evidence for NAADP acting in this early time period (Fig. 6D and fig. S7B).

Fig. 6 RYR1, but not RYR3 or TPC1/2, is the critical Ca

2+ release channel for initial Ca2+ microdomains upon TCR stimulation. (A) Characteristics of initial Ca2+ microdomains in primary WT (n = 68) and Ryr1−/− (n = 31) murine T cells in the first 15 s after bead contact. (B) Subcellular layers at the PM (as indicated) were analyzed in the first second before and after bead contact. Statistical analysis was performed with WT against Ryr1−/− T cells. Mann-Whitney U test across all time points after activation. *P < 0.05, **P < 0.005. (C) Characteristics of initial Ca2+ microdomains formed in the first 15 s after bead contact in primary WT T cells preincubated overnight with dimethyl sulfoxide (DMSO) (n = 13) or with 500 μM BZ194 (n = 34). (D) Subcellular layers at the PM (as indicated) were analyzed in the first second before and after bead contact. (E and F) Characteristics of initial Ca2+ microdomains formed in (E) primary WT (n = 30) and Tpc1−/−/Tpc2−/− (n = 33) T cells and in (F) primary WT (n = 15) and Ryr3−/− (n = 19) T cells in the first 15 s after bead contact. Comparison of the percentage of responding cells, the number of signals per confocal plane per frame, and Ca2+ amplitude (data represent means ± SEM). Statistical significance was determined by Mann-Whitney U test. (G) Colocalization of RYR (green) and ORAI1 (red) in a representative primary WT T cell, showing single optical channels, merge of both optical channels, and colocalization (white). Scale bar, 2 μm. (H) Magnification of a high (1) and a low (2) colocalization area of the ROIs depicted in (G). Scale bars, 100 nm. (I) RYR-ORAI1 plot profile of the arrow shown in image 1 in (H). (J) Statistical colocalization analysis of RYR-ORAI1 (n = 26). Data represent means ± SEM. ****P < 0.0001, unpaired t test.

Because two-pore channels 1 and 2 (TPC1 and TPC2) have been proposed to respond to NAADP (1214), we tested them as well as RYR3 as further candidates regarding potential roles in the formation of spontaneous or antigen receptor–triggered Ca2+microdomains. However, T cells with deletions of both Tpc1 and Tpc2 did not show changes in the percentage of responding cells, the number of Ca2+ microdomains, or their amplitude upon TCR/CD28 stimulation (Fig. 6E). Similarly, genetic deletion of Ryr3 did not alter these Ca2+ microdomain parameters upon TCR/CD28 stimulation (Fig. 6F). We hypothesized that the close functional interaction of RYR1 and ORAI1 proposed here requires colocalization of these proteins, a notion that was confirmed by STED super-resolution microscopy (Fig. 6, G to J). The extent of colocalization between RYR and ORAI1 and STIM1 and ORAI1 in these patches at or very close below the PM was similar, as evidenced by colocalization coefficients of 0.70 ± 0.05 and 0.61 ± 0.03, respectively (Figs. 6J and 3J). It is noteworthy that colocalization of ORAI1, RYR, and STIM1 occurred in some, but not all, regions of the PM (compare Fig. 3H, region 2, and Fig. 6H, region 2), which may be because the most likely sites for such interactions, the ER-PM junctions, cover only a fraction of the PM (15).

DISCUSSION

Here, we identified and characterized spontaneous Ca2+ microdomains below the PM of unstimulated T cells, with amplitudes of 290 ± 12 nM and a frequency of about four signals per second per confocal plane of the cell. These spontaneous Ca2+ microdomains depended on ORAI1 and both STIM1 and STIM2. Preformed complexes of ORAI1 and STIM1 or STIM2 in unstimulated T cells constitute a system to enable continuous low-level Ca2+ entry, resulting in a “basic excitability” of naïve T cells (fig. S8). In the early phase after TCR/CD28 stimulation, a second system consisting of the second messenger NAADP and RYR1 comes into play. Ca2+ release through RYR1 at PM-ER junctions results in an increased number of Ca2+ microdomains, and the concomitant decrease of the luminal Ca2+ concentration of the ER leads to enhanced recruitment and activation of STIM/ORAI1 complexes, thereby further enhancing the formation of Ca2+ microdomains (fig. S8).

Detection of Ca2+ microdomains require a Ca2+ imaging system with high spatial and temporal resolution and a very good signal-to-noise ratio for the Ca2+ signal. The system used here had a detection limit of about 113 nM for Ca2+ amplitude and an image acquisition rate of 40 s−1 at 368-nm spatial resolution, which allowed for detection and characterization of both spontaneous and stimulation-dependent Ca2+ microdomains.

In other cell types, spontaneous Ca2+ microdomains are best studied in cardiac myocytes (16, 17) or astrocytes (18-21). In cardiac myocytes, RYR2 is the major Ca2+ release channel that amplifies low, PM-near Ca2+ signals originating from activation of L-type Ca2+ channels (LTCCs). RYR2 and LTCCs are spatially organized in couplons. In cardiac myocytes from end-stage heart failure patients, spontaneous Ca2+ microdomains are detected in the vicinity of RYR2 localized outside of such couplons, namely, outside the transverse and axial tubular system (16). Analysis of Ca2+ microdomains generated by RYR2 and local sarcomere contractions with second-harmonic generation and two-photon fluorescence microscopy has demonstrated locally coordinated Ca2+ sparks and contractions (17). Although the ER in T cells is not as ordered as the sarcoplasmic reticulum in cardiac myocytes, we observed alternating patches of RYR and ORAI1 at or very close below the PM (Fig. 6, G to J), indicating that proper localization of RYR isoforms may be an important mechanism for regulation of Ca2+ microdomains in general. In astrocytes, spontaneous Ca2+ microdomains of unknown function have been described (18), either as a result of brief openings of the mitochondrial permeability transition pore (19) or by Ca2+ entry across the extracellular space (20), thus mechanistically resembling the system described here for unstimulated T cells. Ca2+ microdomains have been hypothesized to be the local regulatory units of astrocyte interaction with other cell types, such as neurons: Astrocytes release lactate and glutamine to be used as metabolites for neighboring neurons. Such release events appear to be coupled to astrocytic Ca2+ microdomains that (i) locally activate glycolysis, yielding lactate, and (ii) activate enzymes of the citric acid cycle to facilitate adenosine 5′-triphosphate (ATP) formation that can be used for glutamine biosynthesis (21).

In the classical model of capacitative Ca2+ entry, a substantial decrease of luminal Ca2+ concentration in the ER ([Ca2+]lu) is required for ORAI1 activation by STIM1. However, emerging evidence suggests that STIM2 is preclustered close to the PM and activates ORAI1 constitutively, at least in overexpressing cells (2226). Because in unstimulated cells [Ca2+]lu is relatively high, only STIM2, due to its lower affinity for Ca2+, can sense small decreases of [Ca2+]lu that may occur in unstimulated cells, in cells stimulated by low agonist concentrations, or at the very start of stimulation, as in the present study. In this context, STIM2 enhances the sensitivity of ORAI1 activation at relatively high [Ca2+]lu by promoting STIM1 clustering in ER-PM junctions (27). By remodeling the C terminus of STIM1 to an activated conformation, STIM2 traps STIM1 at ER-PM junctions to cause STIM1 coupling to ORAI1, thereby facilitating enhanced Ca2+ entry (28). Accordingly, a model with three different states was proposed: (i) STIM2-ORAI1 clustering at ER-PM junctions at relatively high [Ca2+]lu, (ii) recruitment of STIM1 by STIM2 resulting in STIM2-STIM1-ORAI1 clustering, and (iii) replacement of STIM2 in this ternary complex by STIM1 for full activation of ORAI1 at lower [Ca2+]lu (28). When applying this model to spontaneous Ca2+ microdomains and very early microdomain formation after stimulation in primary T cells, the second state with STIM2-STIM1-ORAI1 clustering appears most likely, because before and in the first 5 s after TCR/CD28 stimulation, single knockouts of Stim2 and Stim1 cause a similar decrease of Ca2+ microdomains as the double knockout (Fig. 5F). Between 5 and about 20 s after TCR/CD28 stimulation, Stim1 knockout gradually shows a less severe phenotype, suggesting that during this phase STIM2 can substitute for STIM1 (Fig. 5F). At later time points (25 to 60 s), the strong dependency on STIM2 also gradually decreases, suggesting that both STIM1-ORAI1 complexes or STIM2-ORAI1 complexes are sufficient to promote Ca2+ microdomains and beginning global Ca2+ signaling. Why STIM2 is able to substitute for STIM1 between 5 and about 20 s after TCR/CD28 stimulation cannot be explained presently and will be the object of future investigation.

In addition to identifying the rapid Ca2+ entry component, we also reanalyzed rapid Ca2+ release involved in Ca2+ microdomain formation. We confirm here RYR1 as the major Ca2+ release channel in this process (Fig. 6, A and B) (8). Furthermore, we demonstrated that NAADP antagonism effectively suppressed antigen receptor–triggered Ca2+ microdomain formation without affecting spontaneous Ca2+ microdomain formation. Together with our data that NAADP does not evoke Ca2+ microdomains in T cells lacking RYR isoforms (8), and with single-channel recordings demonstrating activation of RYR1 by NAADP (29), we hypothesize that NAADP is formed very rapidly and activates Ca2+ release through RYR1 (fig. S8). We have excluded TRPM2 as a potential NAADP target (8). Here, we also excluded other potential NAADP target candidates, TPC1 and TPC2, by demonstrating Ca2+ microdomain formation despite double knockout of these endolysosomal ion channels (Fig. 6E). These results are in accordance with previous findings that TPC1/2 are unrelated Na+ channels activated by phosphatidylinositol 3,5-bisphosphate or regulated in an ATP-dependent fashion by mammalian target of rapamycin (mTOR) (30, 31). Likewise, knockout of RYR3 did not affect Ca2+ microdomain formation in primary T cells (Fig. 6F). However, it is possible that RYR3 plays an important role in Ca2+ signaling at later stages of T cell activation, for instance, in Ca2+-induced Ca2+ release (CICR) and cADPR signaling (2, 32).

Together, we propose a new model for the formation of initial Ca2+ microdomains in T cells (fig. S8). In unstimulated T cells, ORAI1 and STIM1/STIM2 form protein complexes at ER-PM contact sites, resulting in discrete, local Ca2+ entry signals (fig. S8). These constitutive Ca2+ microdomains do not further propagate without cell activation, due to Ca2+ buffering or lowering of [Ca2+]i by Ca2+ adenosine triphosphatases in ER or PM. The preformed ORAI1-STIM1/STIM2 complexes may enable the SOCE machinery in T cells to respond very quickly to TCR stimulation. Preformed ORAI1-STIM1 complexes are reminiscent of the colocalization of these proteins at triad junctions of skeletal muscle fibers (33). Upon TCR stimulation, RYR1 is activated, probably by NAADP formed within a few seconds of T cell stimulation (3), resulting in local and transient Ca2+ release (8). This release contributes directly to Ca2+ microdomains and additionally promotes the activation of STIM1 and STIM2 and thus SOCE through ORAI1 channels within the first second after TCR stimulation. Our findings shed new light on the intricate interaction of RYR1, ORAI1, and STIM1/STIM2, which controls Ca2+ signaling in T cells and thereby adaptive immune responses.

MATERIALS AND METHODS

Reagents

Fluo4-AM, Fura Red-AM, and Fura2-AM were obtained from Life Technologies. Ca2+ indicators were dissolved in DMSO, divided into aliquots, and stored at −20°C until required for use. Anti-mouse CD3 monoclonal antibody (mAb) and anti-mouse CD28 mAb were obtained from BD Biosciences.

Synthesis of BZ194

Under a dry atmosphere of nitrogen, 1.0 ml of bromoacetyl bromide (11.4 mmol, 1 equiv) was dissolved in 25 ml of anhydrous dichloromethane, and the mixture was cooled to −40°C. Under vigorous stirring, 1.9 ml of pyridine (22.9 mmol, 2 equiv) in 15 ml of dichloromethane was added slowly and dropwise. The resulting suspension was stirred for a further 30 min and allowed to warm up to −10°C. Successively, 1.9 ml of n-octylamine (11.4 mmol, 1 equiv) in 15 ml of dichloromethane was added dropwise. The yellow reaction mixture was stirred for 60 min between −10°C and room temperature. Termination of the reaction was carried out by addition of water. The biphasic mixture was diluted further with dichloromethane and washed twice with each 1 M hydrochloric acid (aqueous), sodium bicarbonate (saturated, aqueous), and brine. The organic layer was dried over sodium sulfate and filtrated, and the solvent was evaporated under reduced pressure. The resulting orange oil (2.1 g, 8.31 mmol, 73%) was dried in vacuum, and the obtained 2-bromo-N-octylacetamide was used as such in the next reaction step. In 10 ml of N,N-dimethylformamide, 0.45 g of nicotinic acid (3.7 mmol, 1 equiv) and 0.92 g of 2-bromo-N-octylacetamide (3.7 mmol, 1 equiv) were reacted with one another at 70°C for 24 hours. Successively, the solvent was removed under high vacuum, and the obtained residue was purified by automated normal phase column chromatography using a methanol gradient against dichloromethane (0 to 10% from 0 to 20 min, isocratic from 20 to 30 min). The yellowish solid was crystallized from acetone/methanol, and BZ194 was obtained as white crystals (0.67 g, 1.8 mmol, 49%).

Animal models

The generation of Ryr1−/− (8), Ryr3−/− (34), Tpc1−/−Tpc2−/− (30), Orai1fl/flCd4cre (herein Orai1−/−) (35), Orai2−/−, Orai1fl/flOrai2−/−Cd4cre (8), Stim1fl/flCd4cre (Stim1−/−), Stim2fl/flCd4cre (Stim2−/−), and Stim1fl/flStim2fl/flCd4cre (Stim1−/−/Stim2−/−) (36) has been previously described. WT refers to C57BL/6 mice, and all animals were on a C57BL/6 genetic background. Sex-matched male and female mice between 6 and 13 weeks old were used. Mice were maintained under specific pathogen–free conditions in accordance with institutional guidelines for animal welfare approved by the Institutional Animal Care and Use Committees at New York University School of Medicine, University of Göttingen, University Medical Centre Hamburg-Eppendorf, and the University of Pennsylvania.

Generation of stable transfected Jurkat T cells with G-GECO1.2-Orai1 construct

The G-GECO1.2-Orai1 construct (10) (Addgene plasmid 73562) was a gift from M. Cahalan (University of California, Irvine). The encoding fragment, which was 2.3 kilo–base pairs in size, was excised from plasmid pCMV-OraiG-GECO1.2 by Age I/Mun I digestion and cloned into Age I and Eco RI sites of pF-CAG-GFP (green fluorescent protein) vector (derived from lentiviral pFUGW construct in which the ubiquitin C promoter is exchanged with the chicken actin gene promoter, CAG), replacing GFP open reading frame. Pseudoviral particles were generated by cotransfecting pF-CAG-G-GECO1.2-Orai1 construct with three packaging plasmids (third-generation system) in human embryonic kidney (HEK) 293 cells using a Lipofectamine 2000 reagent (Thermo Fisher Scientific) and culturing in OptiPRO medium supplemented with penicillin/streptomycin and l-glutamine (1:100, Thermo Fisher Scientific) for 48 hours. The supernatant was cleared by centrifugation, filtered through a 0.45-μm filter, and added to Jurkat JMP cells for overnight incubation in the presence of polybrene (8 μg/ml; Sigma-Aldrich). The transduced pool of Jurkat cells was further propagated in RPMI 1640 medium supplemented with 10% fetal calf serum for few passages. To enrich the population for highly expressing cells, the transduced pool was sorted with a FACSAria II flow cytometer (Becton Dickinson) after addition of Tg (Sigma-Aldrich) to a concentration of 2 μM.

Isolation of primary T cells

CD3+ and CD4+ T cells were freshly isolated from spleen and lymph nodes, some of which were shipped to Hamburg, Germany, on wet ice by negative selection using the EasySep Mouse T Cell Enrichment Kit or the EasySep Mouse CD4+ T Cell Enrichment Kit (STEMCELL Technologies Inc.). Cell purity (which was typically 95% T cells) was assessed by immunostaining with fluorescein isothiocyanate–conjugated anti-mouse TCRβ antibody (clone H57-597, BioLegend) and measured with a FACSCalibur flow cytometer (BD Biosciences).

Ca2+imaging in primary T cells

Single-cell Ca2+ imaging was performed as previously described (8). In brief, freshly isolated T cells were loaded with Fluo4-AM (10 μM) and Fura Red-AM (20 μM) for 50 min at room temperature. After washing, the cells were resuspended in Ca2+ buffer [140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes (pH 7.4), 1 mM NaH2PO4, 5 mM glucose]. To stimulate the T cells, protein G beads (Merck Millipore) were coated with antibodies (anti-CD3/anti-CD28) according to the manufacturer’s instructions. Coverslips were coated with bovine serum albumin (5 mg/ml; Sigma-Aldrich) and poly-l-lysine (0.1 mg/ml; Sigma-Aldrich) to facilitate adherence of T cells. Imaging was carried out with a Leica IRBE2 microscope (100-fold magnification) using a Sutter DG-4 as a light source and an electron-multiplying charge-coupled device camera (C9100, Hamamatsu). Exposure time was 25 ms (40 frames/s) in 14-bit mode with twofold binning. A Dual-View module (Optical Insights, PerkinElmer Inc.) was used to split the emission wavelengths with the following filters: excitation (ex), 480/40; beam splitter (bs), 495; emission 1 (em1) 1, 542/50; em2, 650/57.

Fluo4/Fura Red Ca2+image processing

Volocity software (version 6.6.2; PerkinElmer) was used for image acquisition, and Ca2+ image postprocessing was done with Matlab (MathWorks) (37). First, the background in each channel was corrected. Bleaching of Fura Red was corrected using a frame-by-frame elevation of pixel intensities by the difference between the initial value and the fit value of the specific frame with an additive approach. To obtain digital confocal images, Fluo4 and Fura Red were deconvolved using the Lucy-Richardson algorithm with an analytically computed point-spread function, and both fluorescence channels were automatically aligned by a rigid sum-of-squared-differences–based registration. In a final step, the Fluo4/Fura Red ratio was generated, median filtered (3 × 3), and exported.

Ca2+calibration

The initial mean Fura Red fluorescence (Fura Redt0) was as high as the Fura Red fluorescence after correction for bleaching due to the additive frame-by-frame bleaching correction. Thus, the calculation of [Ca2+]i was performed using in situ–determined Rmin (Fluo4min/Fura Redt0 after EGTA chelation) and Rmax (Fluo4max/Fura Redt0) in single-cell measurements. Previously determined mean Kd (dissociation constant) of Fluo4 and Fura Red of 408 ± 12 nM was used (8), and data were subjected to image postprocessing.

Identification of local Ca2+microdomains

Local subcellular Ca2+ microdomains were identified on a frame-by-frame basis and defined as small, compact, connected pixel sets (minimum pixel set size: 6 pixels; maximum pixel set size: 20 pixels; minimum pixel set circularity of 0.5, with circularity defined as [4π × area of pixel set]/[perimeter of pixel set]2) with high [Ca2+]I values compared to the mean [Ca2+]i value in the cell. To distinguish between background noise and Ca2+ microdomains, cell-free homogeneous Ca2+-EGTA buffer ([Ca2+] = 225 nM) containing Fluo4 and Fura Red (8) was analyzed. After processing the cell-free images as described for single-cell images, no noise-induced, fictive Ca2+ microdomains were detected for Ca2+ levels of more than 112.5 nM above the cell-free buffer mean Ca2+ level. Hence, for Ca2+ microdomain detection in cell images, all pixel [Ca2+]i values of the microdomain had to be at least ∆[Ca2+]i = 112.5 nM higher than the frame-specific mean [Ca2+]i of the considered cell. Reported local Ca2+ signal values refer to maximum [Ca2+]i values for the signal pixel set. Data are shown as Ca2+ microdomains per cell and frame.

Ca2+microdomain analysis and comparison for subcellular compartments

To enable analysis and comparison of Ca2+ microdomains for specific subcellular divisions (such as “close to point of activation/bead contact”) and cell groups, a so-called “dartboard projection” approach was introduced: First, a circular, dartboard-like template (with three annuli and individual annuli layers of same area) was matched onto the individual cell images using an automated template-to-cell matching by circle Hough transform (fig. S1). Then, on the basis of their spatial coordinates, the identified local Ca2+ signals were assigned to the corresponding template layers. The resulting dartboards were normalized with respect to the location of the bead contact zone by rotating the template. Last, the cell-specific segment-wise local Ca2+ signal information was aggregated and evaluated for the considered group. All Ca2+ analysis steps were implemented using Matlab.

Preparation of the subcellular fraction P10 and immunoprecipitation of STIM1 and ORAI1

Jurkat T cells (about 2 × 107 lymphocytes per experiment) were harvested, washed in Ca2+ buffer, and homogenized on ice by mechanical disruption in a resuspension buffer (RSB) (pH 7.2; 20 mM Hepes, 10 mM NaCl, 3 mM MgCl2, protease inhibitors). After 20 min of centrifugation at 10,000g, the resulting pellet (P10 fraction) was resuspended in RSB buffer. Protein concentration was assessed by a Bradford reagent (Bio-Rad) protein assay using bovine serum albumin as standard. Four microliters of anti-ORAI1 or anti-STIM1 mAb (Thermo Fisher Scientific) was coupled to protein G magnetic beads (overnight at 4°C). After rinsing the magnetic beads, 400 μg of P10 membranes was used for the pull-down by gently mixing them with 50 μl of bead suspension at 4°C for 1.5 hours. Protein bound to the magnetic beads was collected, washed with phosphate-buffered saline–Tween (0.1%), and resuspended in standard sample buffer for reducing SDS-PAGE (polyacrylamide gel electrophoresis). Protein bound to beads was released by heating (94°C, 10 min), separated by SDS-PAGE (12.5%), and transferred to polyvinylidene difluoride membranes for Western blot analysis (transfer conditions: 14 V, 200 mA, room temperature, 1.5 hours). The blots were immunostained with both primary antibodies (anti-STIM1, 1:1000 dilution; anti-ORAI1, 1:100 dilution), incubated overnight at 4°C. Proteins were visualized with secondary antibodies (incubated for 1 hour at room temperature) coupled with horseradish peroxidase using the SuperSignal West Pico Chemiluminescent (Thermo Fisher Scientific) as the substrate. Experiments with primary T cells were done in a similar manner, except that primary murine T cells were washed in Ca2+ buffer and sonicated on ice (4 × 6 s) to disintegrate the cells. For pull-downs, we used whole-cell lysates from 3 × 107 cells (three spleens) by gently mixing them with 50 μl of bead suspension at 4°C for 1.5 hours.

FRET imaging

Jurkat T cells were transfected with 15 μg of human STIM1 CFP and ORAI1 YFP by electroporation. Both plasmids STIM1 CFP (Addgene plasmid 19755) and Orai1 YFP (Addgene plasmid 19756) were gifts from M. Prakriya (Northwestern University, Chicago) and A. Rao (La Jolla Institute, San Diego) (38). FRET experiments were performed with intact cells 24 hours after transfection. Imaging was carried out with a Leica IRBE microscope (100-fold magnification) using a Sutter DG-4 illuminator as a light source and two electron-multiplying charge-coupled device camera (C9100-13, Hamamatsu). Exposure time was 100 ms in 16-bit mode. A Dual-Cam-View (Optical Insights, PerkinElmer Inc.) was used to split the emission wavelengths with the following filters: CFPex, 430/24; YFPex, 500/20; CFPem, 470/24; YFPem, 535/20. FRET images were corrected for spectral cross-talk (bleedthrough) of the donor and acceptor signal into the FRET channel. Bleedthrough correction was determined by expressing the donor (STIM1 CFP) and acceptor (ORAI1 YFP) separately, and the resulting bleedthrough (CFP, 44%; YFP, 4%) was subtracted from the FRET channel image of double-transfected cells to get the true FRET image. Moreover, 75% of stray light was removed from the FRET images with Openlab’s (version 5.5.2, PerkinElmer Inc.) no-neighbor deconvolution.

STED imaging

Freshly isolated primary T cells were seeded on poly-l-lysine (0.1 mg/ml; Sigma-Aldrich)–coated coverslips. Fixation was done with 3% paraformaldehyde (Electron Microcopy Sciences) for 15 min. In addition, cells were permeabilized with 0.05% (v/v) saponin (Fluka) for 15 min. Primary antibodies [rabbit anti-ORAI1, 1:100 (Proteintech); mouse anti–pan-RYR, 1:100 (Santa Cruz Biotechnology); and mouse anti-STIM1, 1:200 (Thermo Fisher Scientific)] were diluted in 3% (v/v) fetal bovine serum and incubated overnight at 4°C. Secondary antibodies [anti-rabbit STAR RED (Abberior Instruments) and anti-mouse Alexa Fluor 594 (BioLegend)] were incubated (1:200) for 1 hour at room temperature. Coverslips were mounted with Abberior Mount Solid (Abberior) overnight. Images were acquired with the Abberior four-channel easy3D STED with 775 and 595 depletion beams (Abberior Instruments) equipped with a Nikon 60×, 1.4–numerical aperture objective and a QUAD beam scanner. Alexa Fluor 594 was excited with a pulsed 561-nm diode beam, depleted with a pulsed 775-nm STED beam, and detected with a 615 ± 20–nm emission filter. Star red 640 was excited with a pulsed 640-nm diode beam, depleted with a pulsed 775-nm STED beam, and detected with a 685 ± 70–nm emission filter. The pixel size was set to 20 nm. For the colocalization analysis, 40% of stray light was removed with Openlab’s (version 5.5.2, PerkinElmer Inc.) no-neighbor deconvolution. The Manders colocalization coefficient was assessed with the FIJI (version 1.47f) colocalization tool Coloc2. STED imaging was calibrated for xy spatial resolution using fluorescent nanobeads, as detailed in fig. S2.

Ca2+imaging and analysis of Jurkat G-GECO1.2-Orai1 T cells

Jurkat G-GECO1.2-Orai1 T cells were loaded with Fura2-AM (4 μM) for 30 min at 37°C. After rinsing, T cells were resuspended in Ca2+ buffer [140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes (pH 7.4), 1 mM NaH2PO4, 5 mM glucose]. As a control, Jurkat G-GECO1.2-Orai1 T cells were either resuspended in Ca2+-free buffer [140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 20 mM Hepes (pH 7.4), 1 mM NaH2PO4, 5 mM glucose, 1 mM EGTA], incubated for 20 min with 100 μM Synta66 (Aobious) to pharmacologically block CRAC channels, or loaded with 5 μM BAPTA-AM for 30 min. Furthermore, Jurkat G-GECO1.2-Orai1 T cells were incubated overnight with 500 μM BZ194 to specifically block NAADP signaling. Cells were added to prepared coverslips and allowed to adhere before measurement. Imaging was carried out with a Leica IRBE microscope (100-fold magnification) using a Sutter DG-4 as a light source and an electron-multiplying charge-coupled device camera (C9100-13, Hamamatsu). Exposure time was 20 ms for 340 and 380 nm and 400 ms for G-GECO1.2 (488 nm) in 16-bit mode. The following filters were used: ex1, 340/26; ex2, 387/11; ex3, 472/30; bs, 495; em, 520/35. For functional proof of a steady-state mechanism, cells were not stimulated and transient, local Ca2+entry signals were detected using four 10 × 20–pixel ROIs in each cell at the PM after background correction. Tracings of the ROIs were analyzed by determining the F/F0 ratio and signals ≥ ∆ 0.1 ratio units were defined as signal. To test whether the ORAI1-GECO construct worked as expected, at the end of measurements Jurkat G-GECO1.2-Orai1 T cells were stimulated with 1.67 μM Tg (Calbiochem/Merck Millipore).

Statistics

All data are shown as means ± SEM of at least three independent experiments. No specific randomization or blinding protocols were used. Figures were prepared using Matlab and Prism 5 (GraphPad Software). Different groups were compared using a two-tailed unpaired Student’s t test, a Mann-Whitney U test, or a Kruskal-Wallis test using Prism 5. Differences with a P value of <0.05 were considered statistically significant: *P < 0.05; **P < 0.005; ***P < 0.001.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/561/eaat0358/DC1

Fig. S1. Generation of dartboard projection.

Fig. S2. Resolution of the STED microscope.

Fig. S3. Example of an Orai1−/− T cell with Ca2+ microdomains occurring more deeply within the cytosol.

Fig. S4. Markedly decreased initial Ca2+ microdomains in Stim1−/−, Stim2−/−, and Stim1−/−/Stim2−/− T cells.

Fig. S5. Comparison of initial Ca2+ microdomains in primary murine T cells in a dartboard projection.

Fig. S6. ORAI2 is not involved in the formation of initial Ca2+ microdomains.

Fig. S7. Statistical analysis of the formation of initial Ca2+ microdomains in Ryr1−/− and BZ194-treated T cells.

Fig. S8. Model for the formation of initial Ca2+ microdomains in T cells.

Movie S1. Initial Ca2+ microdomains in a WT T cell after stimulation.

Movie S2. Ca2+ responses in an Orai1−/− T cell upon bead contact.

Movie S3. Ca2+ responses in a Stim1−/−/Stim2−/− T cell upon bead contact.

REFERENCES AND NOTES

Acknowledgments: We are grateful to J. Kempski (Hamburg) for help with spleen preparations and R. Fliegert (Hamburg) for help with supplementary movies. We thank M. Prakriya (Chicago) and A. Rao (San Diego) for making available to us expression vectors for STIM1 CFP and ORAI1 YFP via Addgene. Funding: The authors acknowledge financial support by Deutsche Forschungsgemeinschaft [grant GU 360/15-2 (to A.H.G.); SFB 936/A1, Z3 (to F.I.K.); Projektnummer 335447717—SFB1328/A01 (to A.H.G. and A.F.); SFB1328/A02 (to I.M.A.W. and R.W.); SFB1328/A04 (to C.M.); and postdoctoral fellowships KA 4083/2-1 (to U.K.) and VA 882/1-1 (to M.V.)], Landesforschungsförderung Hamburg ReAd Me (project 1 to I.M.A.W. and A.H.G.), Joachim-Herz Foundation (Infectophysics Consortium, project 04, to A.H.G.), and NIH [grants AI097302 and AI107448 (to S.F.) and R01NS055293 and R01NS074257 (to D.R.)]. Author contributions: B.-P.D. contributed to designing this study, carried out experiments, analyzed data, prepared figures, and wrote part of the manuscript. R.W., F.I.K., and D.S. developed and applied software components for image data analyses. P.W., F.C., L.H., C.L., A. Rosche, A.K., U.K., M.V., A.V.F., B.Z., F.I.K., D.L., and A. Ruthenbeck conducted experiments and analyzed data. C.M., A.F., D.R., and S.F. provided reagents, helped plan and interpret experiments, and wrote parts of the manuscript. I.M.A.W. contributed to designing this study, analyzed data, prepared figures, and wrote part of the manuscript. A.H.G. designed the concept of this study and wrote major parts of the manuscript. Competing interests: S.F. is a cofounder of CalciMedica. All other authors declare that they have no competing interests. Data and materials availability: Source data are available upon request from the corresponding author. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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