Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling

Abstract

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca²⁺) influx occurs via highly selective Ca²⁺ release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca²⁺-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca²⁺ levels in areas adjacent to the immune synapse differ from those in the rest of the cytoplasm. Finally, a comparison of phosphorylation patterns of BCR-triggered signaling proteins in the presence or absence of Ca²⁺ revealed the unanticipated finding that initial BCR-triggered, Ca²⁺-dependent tyrosine phosphorylation events involve predominantly Ca²⁺ released from intracellular stores and that influx-derived Ca²⁺ is not essential. This suggests a different role for this phase of Ca²⁺ influx.

INTRODUCTION

Observations made nearly two decades ago suggested that calcium ion (Ca²⁺) is required for antigen receptor-induced activation/signaling processes in B cells (BCR) [1] and that intracellular stores as well as the extracellular environment serve as sources of Ca²⁺ [2]. The amplitude and time course of Ca²⁺ responses as a result of BCR stimulation are important in the activation of certain transcription factors that regulate effector immune functions, cell differentiation, proliferation, or cell death [3, 4]. The majority of work on lymphocyte Ca²⁺ signaling has been focused on T cells, and studies of intracellular Ca²⁺ influx in T cells have shown recently that Ca²⁺ release-activated Ca²⁺ channels (CRAC) comprise the major influx mechanism and that the transmembrane proteins stromal interaction molecule (STIM) and Orai play key roles in this process [5,6,7].

One important feature of the CRAC-mediated mechanism is that intracellular Ca²⁺ concentration increases as a result of influx from extracellular media through store-operated channels (SOC) triggered by a release of relatively small quantities of Ca²⁺ from intracellular stores. There is an ongoing debate about the exact regulatory mechanisms connecting store depletion and opening of extracellular membrane channels. Inositol 1,4,5-triphosphate (IP₃) has been implicated in regulation of membrane channels activity through conformational changes induced by depletion of intracellular Ca²⁺ stores [8].

Several informative studies in B cells (many in avian B cell lines) have been reported [9,10,11], and a number of important signaling molecules have been demonstrated to be involved in the generation of increased intracellular Ca²⁺ in B cells: CD45 [12], CD19 and CD21 [13, 14], STAT3 [15], FcγRIIb [16], Btk [11], acetylcholine [17], B cell linker protein [18], and c-Myc [19]. CD22 has also been shown to play a regulatory role in Ca²⁺ signaling, as BCR-triggered influx is enhanced in CD22-deficient B cells [20]. In addition, a number of BCR-specific features of Ca²⁺ signaling have been described recently with mechanisms involving signal amplification through CD20/CD81, phospholipase Cγ2 (PLCγ2)/IP₃R/STIM1/CRAC, and BCR/cyclic ADP ribose/ryanodine receptor 3/CRAC pathways, as well as modulation pathways that involve CD22, FcγRIIb, SHIP, and Src homology-2-containing tyrosine (Tyr) phosphatases 1/2 (SHP1/2; reviewed in ref. [21]).

Recent studies have also characterized many other important aspects of Ca²⁺ signaling in B cells. It was demonstrated that nonselective cation channels may be involved in BCR-independent Ca²⁺ increases in B cells as a result of shear and osmotic stresses [22], and nonvoltage-gated calcium channels with L-type characteristics can be activated by BCR ligation [23]. Certain SOC properties of B cell Ca²⁺ influx in response to BCR-independent stimulation with thapsigargin (TG) were demonstrated [24]. In addition, other BCR-independent stimuli, such as oxidant stress [25] and peroxide [12], have been shown to elevate intracellular Ca²⁺ in B cells. Lipid raft disruption was found to enhance the release of Ca²⁺ from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux [26]. Syk and Lyn have been demonstrated to play a role in BCR-independent, Ca²⁺-induced apoptosis in B cells [10].

Modulating BCR-mediated Ca²⁺ signaling mechanisms is a promising approach to treatment of B cell-related immune disorders. For example, it has been demonstrated that 1,4-benzodiazepine Bz-423 extends the rise in intracellular Ca²⁺ that accompanies anti-IgM stimulation, and this effect mediates the synergistic death response. As hyperactivation and altered Ca²⁺ signaling are distinguishing features of autoreactive lymphocytes in autoimmune diseases such as lupus, Bz-423 is believed to preferentially target disease-causing cells for apoptosis on the basis of their activation state [27]. Also, Ca²⁺-activated neutral proteases (calpains), which become active in cells responding to signals inducing a rise of cytoplasmic Ca²⁺, are involved in the regulation of apoptosis of some cell types by interaction with caspase-3 and have been shown to play a role in B cell survival [28].

Studies in human B cells that examined the role of extracellular calcium sensing in promoting cell activation [29] have determined that responses to extracellular calcium activated PI-3K/AKT, calcineurin, ERK, p38 kinase, protein kinase C, Ca²⁺/calmodulin kinase II, and NF-κB signaling pathways and resulted in transcription of the early response gene, CD83. This extracellular, calcium-sensing mechanism was also shown to enhance B cell responses to TLR, BCR, and cytokine receptor agonists. These results may indicate a mechanism by which B cells prepare to engage in immune responses by responding to calcium fluctuations in their environment.

However, despite these informative studies, the nature of membrane channels involved in Ca²⁺ influx in mammalian B cells following BCR ligation is still not well understood, and the mechanism of influx has not been characterized to the same extent as in T cells. This report focuses on three major areas of Ca²⁺ signaling in B cells. First, although the role of CRAC in TCR-mediated signaling events is well established [30], the same or a similar mechanism was “presumed” to take place in B cells as well, and several features were identified that linked BCR-triggered Ca²⁺ entry across the plasma membrane to a SOC-type mechanism [21, 31]. However, only limited evidence is available to support this. Our results obtained in primary murine C57BL/6 B cells demonstrate clearly that BCR-mediated Ca²⁺ influx occurs by a store-operated mechanism via highly selective Ca²⁺ release-activated channels and demonstrate the presence and function of a novel mediator molecule involved in the CRAC mechanism, STIM1.

Second, BCR-induced phosphorylation of protein kinases and phosphatases in lymphocytes is thought to precede the increase in intracellular Ca²⁺ concentration, which is the result of influx from the extracellular media [10, 32,33,34,35]. However, the source of Ca²⁺ involved in the initial signaling protein phosphorylation has not been investigated directly. Our results obtained in primary C57BL/6 murine B cells suggest that many initial BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events involve primarily Ca²⁺ released from intracellular stores and do not depend on the extracellular influx.

Third, recent studies have demonstrated an important regulatory role of highly localized signaling events occurring within subcellular structures (microclusters) [36,37,38]. The role of Ca²⁺ in these processes in B cells during antigen response has not been addressed. Our data indicate the presence of dynamic high and low Ca²⁺ areas within the immune synapse as well as larger-scale differences in Ca²⁺ dynamics in bulk cytosol versus synapse area in B cells responding to antigen. These heterogeneities in subcellular Ca²⁺ concentrations are likely indicative of highly localized, Ca²⁺-dependent signaling events taking place in subcellular microclusters.

MATERIALS AND METHODS

Reagents, animals, and cell lines

Ringer’s solution included: 145 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM Hepes, 10 mM glucose, pH 7.4, with NaOH. In 0Ca²⁺ Ringer’s, CaCl₂ was replaced with MgCl₂. K⁺ Ringer’s contained 60 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM Hepes, and 10 mM glucose, pH 7.4, with KOH. 0Ca²⁺ K⁺-Ringer’s contained 60 mM KCl, 1 mM MgCl₂, 5 mM Hepes, and 10 mM glucose, pH 7.4, with KOH. For Mn²⁺ quench experiments, Ringer’s solution was supplemented with 1 mM MnCl₂. For selective ion permeability experiments, 0Ca²⁺ K⁺-Ringer’s was supplemented with 0.1 mM BaCl₂ or SrCl₂. Poly-L-lysine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fura-2-AM, EGTA-AM, BAPTA-AM, 4′, 6-diamidino-2-phenylindole (DAPI), and streptavidin-coated microbeads were from Invitrogen (Carlsbad, CA, USA). Permeabilization with digitonin was performed as described [39,40,41]; AM forms of calcium indicators such as Fura are retained in permeabilized cells significantly better than their non-ester forms (referenced above), and the membrane is first made permeable to divalent cations before it is made permeable to Fura-2-AM, which enables intracellular Ca²⁺ measurements under these conditions. TG was purchased from Alexis Biochemicals (San Diego, CA, USA). C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Intact splenic B cells were purified using the MACS B cell isolation kit (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions. K46μ (μ-chain IgM-expressing) [42] B lymphoma cell line was cultured in IMDM (Gibco, Grand Island, NY, USA), supplemented with 5% FCS (HyClone, Logan, UT, USA), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37°C in 5% CO₂. Antibodies and their sources are as follows: goat anti-mouse IgM F(ab′)₂ and biotin-conjugated goat anti-mouse-IgM F(ab′)₂ (Southern Biotech, Birmingham, AL, USA); donkey anti-mouse IgM Fab/Cy5, normal rabbit, and mouse serums (Jackson ImmunoResearch, West Grove, PA, USA); HRP-conjugated sheep anti-mouse Ig and sheep anti-rabbit Ig (Amersham Pharmacia, UK); anti-phospho (p)Tyr PY20 mAb and anti-pERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-pLyn, anti-pCD19, anti-pSHIP1, anti-pSHP2, and anti-pZap70 (according to the manufacturer, this antibody detects Zap70 only when phosphorylated at Tyr319 but may also cross-react with Syk when phosphorylated at Tyr352; Cell Signaling Technology, Beverly, MA, USA); and anti-mouse B220/allophycocyanin and anti-STIM1 (BD PharMingen, San Diego, CA, USA). Streptavidin was obtained from Biosource (Camarillo, CA, USA).

Intracellular Ca²⁺ measurement

Freshly isolated C57BL/6 B cells or K46μ B cells (2×10⁶ cells/ml in IMDM supplemented with 2.5% FCS) were incubated with Fura-2-AM (1 μM for 30 min at room temperature) and put on a poly-L-lysine-coated, glass-bottom microscopic dish (MatTek, Ashburn, MA, USA) for 15 min, allowing the cells to sediment and adhere. The excess of cells was washed away with normal (or modified as indicated) Ringer’s solution to obtain a monolayer. Cells were left to recover for 10 min to reduce spontaneous Ca²⁺ activity. Time-lapse λ340/380 nm (and 360 nm for Mn²⁺ quench experiments) image acquisition was started, and the dish was perfused with a solution containing stimuli as indicated. Experiments were performed at room temperature. The imaging system was built by Intelligent Imaging Innovations, Inc. (Denver, CO, USA) and uses a Nikon TE2000 microscope with a motorized z-axis-focusing mechanism, Sutter Arc lamp with fiber optical light guide, Cooke SensiCam charged-coupled device camera. We used SlideBook software for image acquisition and analysis and a ratiometric technique for monitoring intracellular Ca²⁺ over time in single cells [43], and single-cell Ca²⁺ traces (typically 100–150 cells) were averaged.

Immunocytochemistry

Cells were adhered to poly-L-lysine-coated coverglass as above, fixed in chilled 4% paraformaldehyde in PBS, permeabilized (0.5% Tween-20, 0.1% FCS, 0.01% saponin in TBS), blocked with SuperBlock (Scytek, Logan, UT, USA), stained with primary antibodies (normal mouse serum for staining controls), followed by fluorescently labeled secondary antibodies, and mounted on glass slides in VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA). Slides were imaged on the microscope system described above. To obtain three-dimensional (3D) models of fluorescently stained cells, images of 30–50 optical serial sections along the z-axis were acquired and reconstructed into a 3D computer model (no neighbors deconvolution; surface rendering algorithms) with SlideBook software (Intelligent Imaging Innovations, Inc.).

Western blot analysis

Cells (2×10⁶/ml) were treated for indicated times with stimuli as indicated and then lysed immediately in 0.5% CHAPS buffer [150 mM NaCl, 10 mM Tris, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 10 mM NaF, 0.4 mM EDTA, 1 mM aprotinin, 1 mM antitrypsin, and 1 mM leupeptin (Sigma-Aldrich), pH 7.5]. Lysates were kept on ice for 30 min and then centrifuged at 10,000 rpm for 10 min at 4°C. Supernatants were mixed with Laemmli SDS-reducing sample buffer (Sigma-Aldrich) and heated for 5 min at 95°C. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized using specific antibodies in conjunction with ECL (PerkinElmer, Boston, MA, USA). Loading controls were done with Ponceau S (BioRad Laboratories, Hercules, CA, USA) staining as follows: Membranes were washed from ECL reagent, incubated with Ponceau S for 10 min, washed again, and scanned for digital densitometry analysis (whole lanes were boxed, and average density was compared between the lanes; obtained values differed by <5%). Densitometry analysis was performed with NIH Image software.

Flow cytometry analysis

Isolated murine splenocytes underwent erythrocyte lysis in red blood cell lysing buffer (Sigma-Aldirch). Cells were washed, stained for extracellular markers (B220), then fixed/permeabilized with Cytofix/Cytoperm solution (Becton Dickinson, San Jose, CA, USA), and blocked with SuperBlock, supplemented with 4% mouse serum. Intracellular staining was performed with mouse anti-STIM1 mAb (BD PharMingen) followed by FITC-conjugated anti-mouse IgG. Population of interest (B220+ B cells) was gated upon, and intracellular staining intensity was analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA). Experiments were performed on a BD LSR flow cytometer (Becton Dickinson).

RESULTS

BCR-triggered Ca²⁺ signaling depends on continuous influx from the outside environment, and the influx is preceded by release of Ca²⁺ from intracellular stores

Removal of extracellular Ca²⁺ after the influx lowers cytoplasmic Ca²⁺ concentration during signaling, indicating that continuous influx from outside is required for sustained signaling. In the experiment presented in Figure 1A, K46μ B cells were labeled with membrane-permeable intracellular Ca²⁺ probe Fura-2-AM and stimulated with 5 μg/ml goat anti-mouse-IgM F(ab′)₂ (anti-IgM further in the text) in Ca²⁺-sufficient media (Ringer’s solution) 200 s after the image acquisition was started. Cells exhibited typical increases in intracellular Ca²⁺. Removal of the extracellular Ca²⁺ at 500 s by a rapid perfusion of the microscopic imaging dish with Ca²⁺-free Ringer’s caused an immediate drop in intracellular Ca²⁺ to the baseline level. An identical result was obtained in primary splenic B cells isolated from C57BL/6 mice (data not shown).

Fig. 1.

B cell Ca²⁺ signaling depends on continuous influx from the extracellular environment, preceded by release of Ca²⁺ from intracellular stores ([Ca²⁺]_i). (A) Removal of extracellular Ca²⁺ during BCR-induced signaling lowers cytoplasmic Ca²⁺ concentration to the baseline level, indicating that continuous influx from outside of the cell is required for sustained Ca²⁺ signaling. (B) Overlapping temporal alignment of the initial Ca²⁺ rise slopes in the presence (solid line) or absence (dotted line) of extracellular Ca²⁺ indicates that the response consists of Ca²⁺ release from intracellular stores, followed, after a delay, by influx from extracellular media; Ca²⁺-free Ringer’s solution was replaced with normal Ringer’s at 1000 s. (C) Mn²⁺ quench data demonstrate a delay between the initial rise in cytoplasmic Ca²⁺ and beginning of its influx through membrane channels: a Ca²⁺-sensitive 340:380 nm ratio (solid line) and Ca²⁺-insensitive:Mn²⁺-quenched isosbestic 360 nm (dotted line) fluorescence of Fura-2. Addition of the stimulus (anti-IgM) is indicated by arrows. Data are representative of three or more independent experiments; each graph is an average of 100–150 individual cell responses. Images were recorded with 10 s intervals.

BCR-triggered Ca²⁺ response consists of initial release of Ca²⁺ from intracellular stores followed, after a delay, by influx from extracellular media

K46μ B cells loaded with Fura-2-AM were stimulated with 5 μg/ml anti-IgM (Fig. 1B) in normal (solid line) or Ca²⁺-free (dotted line) Ringer’s followed by perfusion of the latter with normal Ringer’s at 1000 s. Temporal alignment of averaged Ca²⁺ traces revealed significant overlap on initial stages of the response (<10 s), but the traces diverged at a later time, indicating the effect of an activated influx mechanism. Identical temporal alignment of the initial Ca²⁺ rise slopes in the presence or absence of extracellular Ca²⁺ indicates that the response consists of release of Ca²⁺ from intracellular stores, followed, after a delay, by influx of Ca²⁺ from extracellular media.

Estimating the delay between the release of Ca²⁺ from intracellular stores and start of influx through membrane channels

Mn²⁺ can permeate membrane Ca²⁺ channels and quench the fluorescence of Fura-2 [44]. Measuring Fura-2 fluorescence at its Ca²⁺-independent wavelength (λ360 nm, isosbestic point) in addition to the Ca²⁺-sensitive λ340:380 nm ratio allows monitoring of cytoplasmic Ca²⁺ concentration and influx through membrane channels simultaneously. In experiments presented in Figure 1C, K46μ B cells loaded with Fura-2-AM were stimulated with 5 μg/ml anti-IgM in normal Ringer’s supplemented with 0.5 mM MnCl₂, and fluorescence of Fura-2 excited at λ360 nm (arbitrary units) was recorded simultaneously with the λ340:380 nm ratio. There was no significant change in 360 nm fluorescence in control experiments with nonstimulated cells (<10%; data not shown). Data presented in Figure 1C demonstrate an average delay in the order of ∼84 s between the start of increase in cytoplasmic Ca²⁺ concentration and beginning of Ca²⁺ influx from the outside environment.

Influx in B cells occurs via a capacitative Ca²⁺ entry (CCE) mechanism as a consequence of intracellular stores depletion

TG is known to activate CCE nonspecifically, bypassing receptor-induced generation of IP₃ [30]. As reported, TG-induced CCE Ca²⁺ responses are inhibited when extracellular Na⁺ is replaced by K⁺ (indicating that this Ca²⁺ influx mechanism is not mediated by a Na⁺-Ca²⁺ exchanger pump, as it is impaired as a result of the absence of Na⁺) and blocked in the presence of Ni²⁺ [45]. In our experimental system, B cell Ca²⁺ signaling exhibited all of the above-mentioned features of a CCE mechanism: TG-activated Ca²⁺ influx (Fig. 2A, dotted line); TG-induced Ca²⁺ response was inhibited by replacement of extracellular Na⁺ with K⁺ (Fig. 2A, solid line) and blocked by Ni²⁺ (Fig. 2B; dotted line, without Ni²⁺; solid line, with Ni²⁺). Addition of Ni²⁺ during the ongoing, BCR-triggered Ca²⁺ influx impaired this process significantly, indicating that Ni²⁺ inhibitory activity is not limited only to the initial steps of Ca²⁺ influx (Fig. 2C).

Fig. 2.

Influx in B cells occurs via CCE as a consequence of intracellular stores depletion. BCR-triggered Ca²⁺ signaling exhibits key features of a CCE mechanism: TG activates Ca²⁺ influx (A; dotted line); TG-induced Ca²⁺ response is inhibited by the replacement of extracellular Na⁺ with K⁺ (A; solid line) and blocked by Ni²⁺ (B; dotted line, without Ni²⁺; solid line, with Ni²⁺). Addition of Ni²⁺ during the ongoing BCR-triggered Ca²⁺ influx impairs this process, indicating that Ni²⁺ inhibitory activity is not limited only to the initial steps of Ca²⁺ influx (C). Data are representative of three or more independent experiments; each graph is an average of 100–150 individual cell responses.

Taken together, data presented in Figures 1 and 2 indicate that BCR-triggered transmembrane Ca²⁺ influx occurs via SOC with a short duration between the initial rise from intracellular stores and subsequent extracellular Ca²⁺ influx.

CCE is likely the major ion transport mechanism involved in BCR-induced Ca²⁺ influx

To assess the involvement of non-CCE ion transport mechanisms in BCR-triggered Ca²⁺ influx, we compared the magnitude of Ca²⁺ increases in cells stimulated with anti-IgM and TG versus TG alone. We reasoned that if a BCR-induced influx was activating CCE and non-CCE pathways, the influx in cells stimulated with anti-IgM and TG would be greater than in those stimulated by TG alone as a result of the contribution of a non-CCE mechanism. In these experiments, K46μ cells were stimulated with anti-IgM at 200 s, followed by TG at 500 s (Fig. 3A, dotted line) or in reverse order (TG followed by anti-IgM; Fig. 3A, solid line) in Ca²⁺-free Ringer’s. At 1000 s, the dish was perfused with normal Ringer’s. After the addition of extracellular Ca²⁺, peak values of influx were similar, indicating that CCE is the major mechanism responsible for BCR-mediated Ca²⁺ influx (Fig. 3A). Identical results were obtained with primary B cells isolated from C57BL/6 mice (Fig. 3B). Note that after BCR-mediated release of Ca²⁺ from intracellular stores, TG caused an additional store release (Fig. 3B, upper panel), suggesting that BCR-mediated Ca²⁺ store release does not empty the stores completely.

Fig. 3.

CCE is likely the major ion transport mechanism involved in BCR-induced Ca²⁺ influx. K46μ B cells were stimulated with anti-IgM at 200 s followed by TG at 500 s (A, dotted line; anti-IgM→TG) or in reverse order (TG followed by anti-IgM; A, solid line; TG→anti-IgM) in Ca²⁺-free Ringer’s. At 1000 s, the dish was perfused with normal Ringer’s. An identical result was obtained with purified primary murine B cells (B). After the addition of extracellular Ca²⁺, peak values of influx were similar, indicating that CCE is the major mechanism responsible for BCR-mediated Ca²⁺ influx. Data are representative of four or more independent experiments; each graph is an average of 100–150 individual cell responses.

Ion selectivity of BCR-mediated Ca²⁺ influx

Among other characterized CCE membrane ion channels, CRAC are known to be uniquely selective for Ca²⁺ over other cations [46]. Exploiting the sensitivity of Fura-2 to bivalent cations other than Ca²⁺, we assessed the permeability of ion channels activated as a result of BCR engagement to Ba²⁺ and Sr²⁺. In the experiments presented in Figure 4, K46μ cells were stimulated with anti-IgM in 0Ca Ringer’s at 200 s, which caused the release of Ca²⁺ from intracellular stores, and then at 500 s, the dish was perfused with normal Ringer’s (Fig. 4A, top panel) or Ca²⁺-free Ringer’s supplemented with 2 mM BaCl₂ (Fig. 4A, middle panel) or 2 mM SrCl₂ (Fig. 4A, bottom panel). BCR-activated ion channels were highly selective for Ca²⁺. In control experiments, cells were permeabilized with digitonin in Ca²⁺-free Ringer’s, and then normal, 0Ca + Ba or 0Ca + Sr Ringer’s was added at 500 s. A nonselective influx of ions was detected (Fig. 4B). We acknowledge the possibility of a partial leakage of the Ca²⁺ probe during the experiment (see Materials and Methods); however, the presence of a strong Fura-2-AM fluorescent signal in the permeabilized cells and its sensitivity to changes in Ca²⁺ concentration (see Fig. 6B, upper panel) demonstrate the achievability of intracellular Ca²⁺ measurements under these conditions, at least for the duration of the experiment (∼16 min). Thus, selectivity for Ca²⁺ strongly suggests that BCR-triggered Ca²⁺ influx occurs via the CRAC mechanism.

Fig. 4.

Ion selectivity of BCR-mediated Ca²⁺ influx. K46μ cells were stimulated with anti-IgM in Ca²⁺-free Ringer’s at 200 s, which caused the release of Ca²⁺ from intracellular stores, and then at 500 s, the dish was perfused with normal Ringer’s (A, top panel) or Ca²⁺-free Ringer’s supplemented with 10 mM BaCl₂ (A, middle panel) or SrCl₂ (A, bottom panel). BCR-activated ion channels were highly selective for Ca²⁺. In control experiments, cells were permeabilized with digitonin in Ca²⁺-free Ringer’s, and then, normal, Ca²⁺-free + Ba²⁺ or Ca²⁺-free + Sr²⁺ Ringer’s was added at 500 s. Nonselective influx of ions was detected (B). Data are representative of three or more independent experiments; each graph is an average of 100–150 individual cell responses.

Fig. 6.

Tyr phosphorylation in B cells in the presence or absence of Ca²⁺. (A) K46μ cells were stimulated with 5 μg/ml anti-IgM (a-IgM) F(ab′)₂ for 5 or 15 min in normal or Ca²⁺-free Ringer’s. Whole cell lysates were analyzed by Western blotting with anti-pTyr antibody (PY20). No significant differences in phosphorylation patterns were detected in cells stimulated in Ca²⁺-free or normal Ringer’s. (B) Primary murine B cells (C57BL/6) in 0Ca Ringer’s were or were not (as indicated) pretreated with membrane-permeable Ca²⁺ chelators EGTA-AM (500 μM) or BAPTA-AM (50 μM) to inhibit the effect of Ca²⁺ released from intracellular stores and then stimulated with 5 μg/ml anti-IgM F(ab′)₂ for 5 or 15 min in normal or Ca²⁺-free Ringer’s. Initial BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events involved only Ca²⁺ released from intracellular stores. Data are representative of four or more independent experiments. (C) Phosphorylation of CD19, Lyn, Erk, JNK, Zap70, SHIP1, and SHP2 in the presence or absence of Ca²⁺. Only Ca²⁺ released from intracellular stores was required for the phosphorylation of these signaling molecules. Primary murine C57BL/6 B cells were stimulated as in B. Whole cell lysates were analyzed by Western blotting with specific anti-phospho antibodies as indicated. Data are representative of three or more independent experiments.

Overall, data presented above (Figs. 1234) provide direct evidence that CRAC is the major mechanism of intracellular Ca²⁺ influx in B cells upon BCR ligation with antigen.

Ca²⁺ signaling peak precedes the formation of central super molecular activation cluster (cSMAC)

The immune synapse is an important functional structure involved in the regulation of antigen receptor complex signaling in B cells. However, the temporal relation between Ca²⁺-dependent signaling events and formation of the immune synapse in B cells has not been clearly established. Data presented in Figure 5, A and B, demonstrate that the peak of intracellular Ca²⁺ influx occurs before the formation of the synapse. In these experiments, K46μ cells were labeled with Cy3-conjugated anti-IgM Fab (monovalent-nonstimulating) and placed on a glass-bottom microscope dish coated with unlabeled anti-IgM F(ab′)₂ [47]. Immune synapse formation was monitored simultaneously with intracellular Ca²⁺ (Fig. 5A). Quantified Ca²⁺ dynamics in the same cell is presented in Figure 5B. Peak Ca²⁺ signaling (100–600 s) occurred before the formation of a distinct central cluster of BCR molecules (cSMAC; after ∼900 s).

Fig. 5.

(A) Immune synapse formation (BCR clustering) in K46μ B cells responding to anti-BCR stimulus immobilized on glass. K46μ cells were labeled with Cy3-conjugated anti-IgM Fab (monovalent) and placed on anti-IgM F(ab′)₂-coated glass. Immune synapse formation was monitored simultaneously with intracellular Ca²⁺. (B) Quantified intracellular Ca²⁺ dynamics in the same cell: peak Ca²⁺ signaling (100–600 s) occurred before the formation of a distinct central cluster of BCR molecules (∼900 s). Data are representative of six independent experiments (a total of 36 single cells was analyzed). (C) Representative examples of cytoplasmic Ca²⁺ gradients in K46μ B cells responding to a polarized stimulus. In these experiments, anti-IgM-coated beads (outlined in purple) were brought into contact with K46μ cells loaded with Fura-2-AM and adhered to the bottom of a microscopic dish (in normal Ringer’s). Static images (λ340:380 nm ratio; random time-points, <20 min) demonstrate distinct high and low Ca²⁺ areas within the cytoplasm, probably reflecting different stages of activation/Ca²⁺ signaling in the immune synapse area (beads were contacting B cells at random; therefore, their interaction times with a particular cell were different).

Cytoplasmic Ca²⁺ heterogeneities in areas adjacent to immune synapse

There is not an established understanding of the localization of Ca²⁺ influx channels in lymphocytes during the initial antigen recognition and signaling [30, 43, 46], and the distribution of Ca²⁺ channels on B cell membrane during the formation of immune synapse has not been addressed. Our experiments identified subcellular Ca²⁺ gradients, indicating that membrane Ca²⁺ channels in B cells may preferentially relocate (or be more active without relocation) to the area of the cell contacting a corpuscular/directional stimulus. Representative examples of cytoplasmic Ca²⁺ gradients in K46μ B cells responding to a polarized stimulus (anti-IgM immobilized on microbeads) are shown in Figure 5C. In these experiments, anti-IgM-coated beads (streptavidin-coated beads incubated with biotin-conjugated anti-IgM and then washed) were brought into contact with K46μ cells (loaded with Fura-2-AM and adhered to the bottom of a microscopic dish in normal Ringer’s). Static images (λ340:380 nm ratio; random time-points, <20 min) demonstrate distinct high and low Ca²⁺ areas within the cytoplasm, possibly reflecting different stages of activation/Ca²⁺ signaling in the immune synapse area (beads were contacting B cells at random; therefore, their interaction times with particular cells were different).

BCR-mediated phosphorylation of signal transduction proteins in the presence or absence of Ca²⁺

Tyr phosphorylation is one of the major processes taking place during the activation of the cell in response to external stimuli, and the presence of Ca²⁺ is important in this process. We have examined BCR-mediated phosphorylation of signal transduction proteins in B cells stimulated in the presence or absence of Ca²⁺ to identify the source of Ca²⁺ involved in BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events (intracellular stores vs. influx). K46μ cells were stimulated with 5 μg/ml anti-IgM for 5 or 15 min in normal or Ca²⁺-free Ringer’s solution (Fig. 6A). Whole cell lysates were analyzed by Western blotting with anti-pTyr antibody (PY20). No significant differences in phosphorylation patterns were detected in cells stimulated in 0Ca²⁺ or normal Ringer’s. In addition, we assessed dose-response properties of pTyr in B cells stimulated with anti-IgM at different concentrations in the presence of absence of extracellular Ca²⁺ (Supplemental Fig. 1). The results demonstrate that B cells remain capable of BCR-induced pTyr responses through a wide range of stimulus doses regardless of trasmembrane Ca²⁺ influx. In Figure 6B, primary murine C57BL/6 B cells were pretreated for 20 min (followed by wash) with membrane-permeable Ca²⁺ chelators (500 μM EGTA-AM or 50 μM BAPTA-AM) in Ca²⁺-free Ringer’s to inhibit the effect of Ca²⁺ released from intracellular stores upon BCR engagement. No significant increase in Tyr phosphorylation was detected in cells with EGTA-AM or BAPTA-AM. Control experiments demonstrated that effects of these Ca²⁺ chelators on B cells were reversible and did not exhibit toxic effects. In these experiments, chelator-treated B cells (K46μ) were placed in high calcium buffer for 30 min [Ringer’s solution supplemented with 20 mM CaCl₂ (normally 2 mM)] to allow efficient saturation of the chelator. Cells were then washed with normal Ringer’s solution, and Ca²⁺ responses to anti-IgM stimulus in chelator-treated cells were not significantly different from those of untreated control cells (data not shown).

These results suggest that initial BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events involve mostly Ca²⁺ released from intracellular stores and that its influx from outside is not critical at early signaling stages. In addition, we have assessed the role of Ca²⁺ in phosphorylation of several signaling molecules known to play major roles in B lymphocyte signal transduction (CD19, Lyn, Erk, JNK, Zap70, SHIP1, and SHP2). Figure 6C represents a series of Western blot experiments with primary murine B cells (C57BL/6) stimulated in the presence or absence of extra- and intracellular Ca²⁺, similar to the experiment shown in Figure 6B. None of the tested signaling proteins demonstrated detectable increases in phosphorylation levels when intracellular stores Ca²⁺ was blocked by EGTA-AM or BAPTA-AM, and no significant differences in activation were found in the presence or absence of extracellular Ca²⁺.

Ca²⁺ signaling protein STIM1 is present in B cells and is involved in BCR-triggered intracellular Ca²⁺ influx

To further understand the mechanisms of B cell Ca²⁺ influx, we confirmed the presence of STIM1 in C57/BL/6 primary murine B cells and in our model cell line by fluorescent microscopy, flow cytometry, and Western blotting. First, intracellular staining with anti-STIM1 mAb in permeabilized primary murine B cells has demonstrated that STIM1 is present (Fig. 7A, flow cytometry analysis). STIM1 was also detected by Western blotting in whole cell lysates of the K46μ murine B cell line used in many experiments in this study and in the BCR-expressing human RL B cell line (American Type Culture Collection #CRL-2261) used as a control (Fig. 7B). Noteworthy, STIM1 in K46μ B cells appeared in what is likely to be two different isoforms, as two distinct bands of different molecular weights were detected specifically with anti-STIM1 mAb. In addition, intracellular localization of STIM1 was confirmed by immunofluorescent microscopy (Fig. 7C; upper panels, 2D images; lower panels, 3D reconstruction of multiple images acquired along the z-axis). The presence of this novel Ca²⁺ regulatory protein, known to be involved in CRAC mechanism, is consistent with the possibility for a STIM-mediated CRAC mechanism in B cells, similar to the one described in T cells. Our results also indicate that STIM1 is involved in antigen receptor-induced Ca²⁺ signaling in B cells, as inhibition of STIM1 expression with specific siRNA (Dharmacon, Inc.) reduced levels of BCR-triggered Ca²⁺ influx substantially (Fig. 7, D and E). Furthermore, B cells with reduced STIM1 levels had essentially unaltered pTyr levels in response to anti-BCR stimulus (Supplemental Fig. 2), which further supports our conclusion that intracellular stores provide a major source of Ca²⁺ involved in BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events.

Fig. 7.

Calcium signaling protein STIM1 is present in B cells. (A) Intracellular staining with anti-STIM1 mAb followed by FITC-conjugated secondary antibody in permeabilized C57BL/6 primary murine B cells. (B) Western blot of whole cell lysates from C57BL/6 and K46μ murine B cells; RL human B cells served as a control. STIM1 appeared in two different isoforms in K46μ cells. (C) Intracellular localization of STIM1 visualized by fluorescent microscopy (upper panels, 2D images; lower panels, 3D reconstruction of multiple images acquired along the z-axis; RL B cell is shown); DAPI (blue, nucleus); STIM1 (green); surface BCR (IgM; red). (D) B cells (K46μ) were transiently transfected with murine STIM1 small interfering (si)RNA using the RNA transfection kit and protocol (Dharmacon, Inc., Lafayette, CO, USA). After 48 h, STIM1 protein levels were reduced significantly, as assessed by Western blotting with the anti-STIM1 antibody (upper panel); loading control with anti-GAPDH antibody (lower panel). Control cells were treated with the transfection reagent to control for toxic effects; results are shown in triplicates. (E) STIM1 siRNA-treated cells described in D exhibited diminished levels of Ca²⁺ influx in response to anti-IgM stimulus; data are the average of 169 (control) and 144 (STIM1 siRNA) single B cell intracellular Ca²⁺ traces. Experiments were performed in triplicates.

DISCUSSION

This study has characterized several important and previously undefined features of antigen receptor-triggered Ca²⁺ signaling in B lymphocytes. Our results, obtained in C57BL/6 primary murine B cells and the BCR-expressing model cell line K46μ, provide a direct line of evidence that BCR-mediated Ca²⁺ influx occurs by a store-operated mechanism via highly selective CRAC. We demonstrated that BCR-triggered Ca²⁺ signaling depends on continuous influx from the outside environment, is preceded by the release of Ca²⁺ from intracellular stores, and consists of the initial release of Ca²⁺ from intracellular stores, followed, after a delay, by influx from extracellular media. These data also indicate that intracellular signaling events preceding the activation of membrane Ca²⁺ channels are not dependent on the presence of extracellular Ca²⁺. With Mn²⁺ quench approach measuring Fura-2 fluorescence at a Ca²⁺-independent isosbestic point, we estimated an average delay in the order of ∼84 s between the release of Ca²⁺ from intracellular stores and the start of influx through membrane channels. Taken together, these results (Fig. 1) indicate that BCR-triggered transmembrane Ca²⁺ influx occurs through SOC. In a series of experiments with a nonspecific activator of CCE TG, which nonspecifically activates CCE bypassing receptor-induced generation of IP₃ [30], we have demonstrated that influx in B cells occurs via a CCE mechanism as a consequence of intracellular stores depletion. Consistent with other studies performed in T cells, TG-induced CCE Ca²⁺ responses in our experimental system were inhibited by replacement of extracellular Na⁺ with K⁺ (indicating that this Ca²⁺ influx mechanism is not mediated by a Na⁺-Ca²⁺ exchanger pump) and blocked in the presence of Ni²⁺ [45] (Fig. 2). Combined application of these stimuli (anti-IgM+TG) demonstrated that CCE is a primary ion transport mechanism involved in BCR-induced Ca²⁺ influx; hence, BCR-induced influx does not activate any other/non-CCE pathways, as the influx in cells stimulated with anti-IgM + TG was not greater than in those stimulated by TG alone, which would have reflected the contribution of a non-CCE mechanism (Fig. 3). Our results also revealed that BCR-mediated CCE is uniquely selective for Ca²⁺ over other cations (Fig. 4), which is a characteristic feature of CRAC [46]. Overall, these results (Figs. 1234), for the first time to our knowledge, provide a compilation of direct evidence that CRAC is the major mechanism of intracellular Ca²⁺ influx upon BCR engagement in mammalian B cells. However, electrophysiological properties of intracellular CRAC (I_crac) in primary murine B lymphocytes are beyond the scope of this study. Imaging experimental approaches for measuring intracellular calcium have been proven in studies of CRAC in T lymphocytes [30, 48, 49].

STIM1 is a novel transmembrane Ca²⁺ regulatory protein recently identified and implicated to play a key role in CRAC-mediated Ca²⁺ influx in T cell lines [7, 50]. The role of STIM1 in antigen receptor-mediated Ca²⁺ signaling in primary murine B cells and its subcellular localization has not been studied in detail. Although the presence of STIM1 in B cell lines has been suggested earlier [51, 52], the results were preliminary and not focused on B cells. Most of the current knowledge about STIM comes from non-B cell studies, including insect cells, fibroblasts, and T cells. This mechanism is believed to be evolutionary conserved and to include B lymphocytes, whereby STIM directly monitors the Ca²⁺ concentration in the endoplasmic reticulum (ER) lumen. These possibilities are discussed in an excellent review [53] along with potential new roles for distinct Ca²⁺mobilization profiles in individual primary B cell subsets and nuclear translocation of transcription factors as downstream targets of Ca²⁺ mobilization.

Recent mutant analysis findings based on structure–function experiments in chicken DT40 cells suggested that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the plasma membrane and activation of SOC channels [54]. It has also been demonstrated that the coupling of Ca²⁺ store release to I_crac channel activation in DT40 chicken B cells requires tonic activity of Lyn and Syk kinases and that the action of kinases on I_crac activation does not arise from control of the expression level of STIM1 and Orai1 proteins [55]. Smyth et al. [56] examined Ca²⁺ store-dependent reversal of STIM1 localization in human embryo kidney 293 cells and demonstrated that SOC Ca²⁺ entry is tightly coupled to formation of STIM1 puncta, and SOC and puncta formation involve a dynamic and reversible signaling complex. Expression of STIM1 and STIM2 has been reported in CD3+/CD4+, CD3+/CD8+, and CD19+ murine lymphocytes [57]. However, recent reports suggest that unlike STIM1, STIM2 has a smaller role in T lymphocyte signaling [58]. Given a considerable level of similarities between BCR and TCR signaling mechanisms, STIM2 may not play a major role in B cells as well.

Our data clearly confirm the presence of STIM1 in C57/BL/6 murine B cells as well as in our model cell lines, as assessed by fluorescent microscopy, flow cytometry, and Western blotting (Fig. 6C), and its function in BCR-triggered intracellular Ca²⁺ influx as inhibition of STIM1 expression with specific siRNA reduced the influx substantially (Fig. 7, D and E). This provides additional evidence in support of our conclusion that CRAC is a major mechanism of antigen receptor-mediated Ca²⁺ influx in B cells.

A number of studies focused of Ca²⁺ transport and signaling in B cells with a special emphasis on the PLCγ2-IP₃-mediated Ca²⁺ signaling mechanism triggered by BCR and its coreceptors (CD19, CD21), as well as other mechanisms involved in the regulation of cytosolic Ca²⁺ concentration. It has been discussed how these combined inputs could have an impact on the Ca²⁺-dependent regulation of NFAT and NF-κB transcription factor pathways and influence cell-fate choice during humoral immune responses (reviewed in ref. [59]). Different B cell subsets may differentially modulate Ca²⁺ signaling to control B cell fate, and our previous findings demonstrated substantial differences in Ca²⁺ responses triggered by the engagement of BCR [13] as well as BCR coreceptors such as CD21 [60] in B cell subsets (mature, immature, marginal zone, B-1/B-2 cells) and effects on B cell development. Our recent results [61] have also indicated that CD21-enhanced Ca²⁺ signaling plays a major role in overcoming B cell anergy and triggering antigen-specific antibody responses in a subset of anergic B cells.

The immune synapse is an important regulatory structure in B cells that plays a key role in antigen presentation/processing, interactions between different cells of the immune system, determining signaling thresholds through the engagement of coreceptors, and regulating localized increases of antigen density on the cell surface. Our studies of the relationship between Ca²⁺ signaling and immune synapse dynamics have demonstrated that the Ca²⁺ signaling peak precedes the formation of cSMAC by an estimated 300 s (Fig. 5, A and B). This is consistent with other studies that found immune synapse playing a down-regulatory role in NK and T lymphocyte activation after the initial receptor engagement [62, 63].

Furthermore, our experiments have revealed distinct synapse-related cytoplasmic Ca²⁺ gradients (Fig. 5C): Ca²⁺ levels in areas adjacent to the immune synapse were different from those in the rest of the cytoplasm, indicating that membrane Ca²⁺ channels in B cells may preferentially relocate (or be more active without relocation) to the area of the cell contacting a corpuscular/directional stimulus, probably reflecting different stages of activation and Ca²⁺ signaling in the immune synapse area (i.e., stimulus-coated beads were contacting B cells at random, and their interaction times with particular cells were different). It is our interpretation that at the time the still image was taken (Fig. 5C), those beads that arrived earlier had already gone through initial stages of immune synapse formation accompanied by localized cytoplasmic Ca²⁺ increases and therefore, had selectively lower cytosol Ca²⁺ in the synapse area. In contrast, the beads that arrived later were at earlier stages of interaction with the cell, as indicated by higher Ca²⁺ levels in the area adjacent to the contact site. This observation also suggests the presence of a highly localized mechanism that enables compartmentalized regulation of Ca²⁺ levels within the cytoplasm, and as a result, a cell is able to process several corpuscular antigenic stimuli simultaneously and independently. This mechanism may have important implications to “multifaceted” responses, whereby a B cell that is not terminally differentiated is able to balance multiple stimuli of different antigen specificities (or affinities/avidities) and eventually select a “preferred” stimulus to develop a specific response (based on BCR signal strength and timing, contributions from other signaling pathways introduced by the involvement of coreceptors and other factors). A possible caveat to these observations is that intracellular Ca²⁺ gradients may appear as an imaging artifact as a result of intracellular granules having Ca²⁺ levels different from those in the bulk cytosol, as was described in cytotoxic T lymphocytes rich with acidic lytic granules [43]. However, B cells are not likely to contain nearly as much acidic granules as cytotoxic T cells. Furthermore, imaging artifacts that may appear as intracellular Ca²⁺ gradients as a result of different Ca²⁺ levels in the nucleus, intracellular granules, or other organelles ought to be distributed randomly (i.e., without any relation to the immune synapse site), which was not the case in our experiments (Fig. 5C). Noteworthy, studies performed in cytotoxic T cell lines demonstrated that Ca²⁺ channels do not cluster in the synapse area and are distributed uniformly throughout the cell surface during the first few seconds of signaling [43]. Our data, however, were collected at much later time-points (minutes into the synapse formation), which may account for the differences in the results. In addition, we cannot completely rule out other unanticipated imaging artifacts that may result from selective partitioning of the dye or uneven Ca²⁺ buffering.

The dynamic high and low Ca²⁺ areas within the immune synapse as well as larger-scale differences in Ca²⁺ dynamics in bulk cytosol versus synapse area in B cells responding to a localized stimulus are indicative of highly localized Ca²⁺-dependent signaling events taking place in subcellular microclusters. One potential experimental approach to investigate these findings further would rely on the development of calmodulin-cyan fluorescent protein/yellow fluorescent protein fluorescence resonance energy transfer-based Ca²⁺ sensors [38] targeted to molecules that participate in formation of the immune synapse in B cells (Igα/β, CD19, CD21). Such molecule-targeted biosensors can be used for real-time monitoring of Ca²⁺-dependent signaling events in the immediate proximity of molecular signaling microsites. In addition, the heterogeneities in BCR-triggered subcellular Ca²⁺ levels are likely to correlate with localized, Ca²⁺-dependent Tyr phosphorylation events. These possibilities are currently under study in our laboratory.

Antigen receptor-induced phosphorylation of protein kinases and phosphatases is required for activation of the BCR-mediated Ca²⁺ signaling pathway. In the absence of kinase phosphorylation, molecular pathways linking BCR ligation/activation to IP₃ production are not functional, as the phosphorylation and activation of PLCγ, which results in IP₃ production, do not occur (reviewed in ref. [64]). In general, pTyr activity in lymphocytes is thought to precede the increase in intracellular Ca²⁺ concentration [10, 32,33,34,35], which is the result of influx from extracellular media through SOC triggered by the release of relatively small quantities of Ca²⁺ from intracellular stores. Our results, however, indicate that many initial BCR-triggered Ca²⁺-dependent Tyr phosphorylation events involve only Ca²⁺ released from intracellular stores and do not depend on influx from the outside environment (Fig. 6, A and B). Control experiments demonstrated that treatment with EGTA/BAPTA-AM did not severely deplete intracellular Ca²⁺ levels in the cells (data not shown), although chelator-treated cells tended to have somewhat lower Ca²⁺ levels. Other studies have also demonstrated that treatment with membrane-permeable chelators such as EGTA-AM or BAPTA-AM does not deplete cytoplasmic Ca²⁺ significantly below physiological levels [43]. Consistent with our interpretation, recent studies [65] have demonstrated the link between Ca²⁺ and signaling protein phosphorylation, in particular, that the Ca²⁺ and reactive oxygen intermediates generated upon BCR activation can engage in a cooperative interaction that amplifies early signaling events.

As a discovery-based, preliminary approach, we have attempted to assess the role of store- versus influx-derived Ca²⁺ in phosphorylation of several major signaling proteins involved in BCR signaling using a conventional Western blotting technique and a small panel of phosphor antibodies (Fig. 6C), including Zap70, which was described recently in B cells [66]. At this time, we were unable to identify specific signaling molecules that become phosphorylated distinctly before or after the initiation of Ca²⁺ influx. A large-scale phosphoprotein array analysis is more appropriate for this purpose, and this possibility is currently under study in our laboratory.

The presence of two distinct types of Ca²⁺-mediated protein phosphorylation events (those that use Ca²⁺ released from intracellular stores and events relying on Ca²⁺ brought in by the transmembrane influx) raises a possibility for the following previously unanticipated regulatory role of Ca²⁺ in B cell activation. The initial Ca²⁺ increase that results from the depletion of intracellular stores is approximately tenfold lower than the increase caused by transmembrane influx (Figs. 3 and 4). We speculate that low store-derived Ca²⁺ levels primarily accompany only the initial signaling events that are involved in cell activation, and the significantly higher post-influx Ca²⁺ levels that occur later may also play a role in the inhibitory signaling cascades that down-regulate activation mechanisms.

Overall, this study has provided several important, novel insights into the role of Ca²⁺ in B cell signal transduction. We provided cumulative evidence that highly selective CRAC comprise the major mechanism of intracellular Ca²⁺ influx in mammalian B cells upon BCR engagement. Noteworthy, these results were obtained in a series of coherent experiments performed in one experimental system, which further validates our results compared with reports published previously that were focused only on several particular aspects of Ca²⁺ influx mechanisms in various B cell models (primarily avian B cell lines). We have also confirmed that a novel CRAC regulatory molecule STIM1 is present in B cells and plays an important signaling role in Ca²⁺ signaling. We have identified a temporal relation between Ca²⁺-dependent signaling events and formation of the immune synapse in B cells. Our findings have also revealed localized differences in cytoplasmic Ca²⁺ levels within the immune synapse area, possibly reflecting different stages of signaling and progression in synapse formation. Finally, we have demonstrated that initial BCR-triggered, Ca²⁺-dependent Tyr phosphorylation events involve mostly Ca²⁺ released from intracellular stores and that its influx from outside is not critical at these stages of B cell activation.