Introduction

Ca2+ is a ubiquitous intracellular messenger that encodes a plethora of cellular functions in response to agonists or environmental stimuli. These include contraction, gene transcription, secretion, cellular proliferation, and apoptosis to name a few. Given the disparate often conflicting (cell death vs proliferation for example) cellular functions determined by Ca2+ signals, cells have developed mechanisms to encode specific cellular responses in the spatial, temporal and amplitude dynamics of Ca2+ signals. Spatial encoding can be enforced by co-localization of the Ca2+ source (typically a Ca2+ channel) and the target effector. Ca2+ diffusion around the Ca2+ source is restricted by physical and biochemical barriers, creating a space and time limited microdomain where Ca2+ can effectively and specifically activate a target. The microdomain can range from a few nm (nanodomains) to a couple of µm depending on multiple factors, including but not limited to channel density and conductance and buffering capacity in the microdomain1. Stimulating a Ca2+ sensitive mechanism far from the main Ca2+ source is consequently a signaling challenge since Ca2+ needs to be carried to the effector while avoiding non-specific activation of Ca2+ effectors along the way2.

Ca2+-linked agonists mobilize store Ca2+ release typically through activation of IP3-Receptors (IP3Rs), leading to store depletion and to the opening of Store-Operated Ca2+-Entry (SOCE) channels at the plasma membrane (PM). SOCE activation in response to ER store depletion, is mediated by the ER transmembrane Ca2+ sensors of the STIM family (STIM1 and STIM2 and their splice variants), which undergo a conformational change in response to reduced luminal Ca2+ concentrations, leading to their clustering at ER-PM contact sites (MCS), and recruitment and gating of the PM Ca2+ channels of the Orai family (Orai1, 2 and 3)3. Ca2+ flowing through Orai1 channels is subsequently taken up into the ER by the Sarcoplasmic Endoplasmic Reticulum ATPase (SERCA) pump, thus refilling Ca2+ stores and preparing the cell for another round of signaling. However, in addition to store refilling SOCE specifically activates multiple downstream effectors3,4,5. A limitation in that regard is the spatial spread of the SOCE Ca2+ microdomain. The assembly of STIM and Orai is not diffuse at the plasma membrane, but highly localized to ER-PM MCS where the two proteins form dense clusters visible at the plasma membrane plane and enhanced in size when SOCE elements are overexpressed6. Accumulating evidence suggests that these clusters are not restricted to a simple STIM/Orai partnership but contain numerous additional proteins, including the SERCA pump. Several studies report that, upon store depletion, SERCA localizes to the SOCE clusters5,6,7,8,9,10. This increases the efficiency of Ca2+ store refilling by placing the pump close to SOCE Ca2+ entry source. This particular organization of the SOCE complex defines a spatial Ca2+ microdomain that limits the spread of Ca2+ ions entering through Orai1 to a limited sub-membrane area6. The actual size of this domain is still a matter of debate. The cell type, the extent of store depletion and the physiological state of the cell will generate various cluster sizes. In addition, experimentally STIM-Orai clusters have been measured following overexpression, which is likely to influence their size6. Nonetheless, the most accurate estimates of SOCE clusters come from thin sections studies where the maximal diameter is around 300 nm11,12. These clusters are also tightly confined in the “z-axis” due to the close apposition of the ER and PM to create a “disc-shaped” microdomain with a 10–20 nm in thickness6,13. This means that for downstream targets to be specifically responsive to SOCE they have to localize in this spatially restricted and cluttered microdomain. This is the case for adenylate cyclase 8 and for the Ca2+-activated phosphatase calcineurin, which couples to Orai114,15.

Another mechanism to transport Ca2+ in the long-range and expand the SOCE Ca2+ microdomain that has been well described in polarized pancreatic acinar cells and Xenopus oocytes is Ca2+ tunneling7,16. Ca2+ tunneling involves the transport of Ca2+ ions that flow through SOCE channels into the ER lumen, followed by their release through IP3 receptors at distal sites to activate effectors without inducing a global Ca2+ rise (see recent review2). Here we extend the characterization of Ca2+ tunneling and show that it encodes exquisite specificity in activating effectors downstream of SOCE. Using real time simultaneous imaging of Ca2+ in the ER lumen, cytosol and mitochondria, we show that Ca2+ tunneling differentially signals to downstream effectors. Whereas, Ca2+ influx through Orai1 within the SOCE microdomain effectively activates NFAT translocation, Ca2+ tunneling expands the spatial spread of SOCE by activating other PM effectors such as Ca2+-activated potassium channels. Surprisingly however, tunneling is unable to mediate a Ca2+ rise in the mitochondria arguing that in the non-polarized HeLa cell it results in a spatially restricted cytosolic Ca2+ rise in the cell cortex. This indicates that the long range tunneling mechanism described in polarized cells has been adapted to a preferentially cortical Ca2+ signaling module downstream of SOCE allowing selective activation of Ca2+-dependent effectors.

Results

Simultaneous real time Ca2+ imaging in the cytosol, ER and mitochondria

We initially sought to determine the response of mitochondria to Ca2+ tunneling downstream of SOCE. Conceptually mitochondria represent an ideal target for Ca2+ tunneling, as they form close membrane contact sites with the ER (Mitochondria Associated Membranes or MAMs) where IP3 receptors are enriched17. Furthermore, the mitochondria are a major target of Ca2+ signaling, and play a critical role in buffering cytosolic Ca2+ transients. The relationship between SOCE and the mitochondria is complex and exhibits some cell specific nuances. In immune cells the mitochondria have been shown to act as a Ca2+ sink for Ca2+ flowing through SOCE, which in turn modulates its function18,19,20. The mitochondria is further well established as a low pass filter for cytosolic Ca2+ signals and have been shown to functionally interact with SOCE and modulates its signaling21. Furthermore, the subcellular localization of mitochondria affects their response differentially to the source of Ca2+. In both HeLa cells and pancreatic acinar cells the source of Ca2+ was shown to differentially stimulate different populations of mitochondria based on subcellular localization22,23. In HeLa cells mitochondria localize away from the plasma membrane24, making them an interesting distal target for Ca2+ tunneling.

To test whether the mitochondria act as an effective downstream effector of Ca2+ tunneling, we imaged Ca2+ dynamics simultaneously in the mitochondria, ER lumen and cytosol. A recently developed family of Ca2+ sensors termed CEPIAs (Ca2+-measuring organelle-Entrapped Protein Indicators), can be targeted to the ER or to the mitochondria, and allow simultaneous imaging of the two compartments25. We thus combined R-CEPIAer (ER lumen Ca2+ sensor) with G-CEPIA2mt (mitochondrial Ca2+ sensor) to image Ca2+ changes in the ER and mitochondria respectively, because their spectral properties allow for imaging cytosolic Ca2+ as well using Fura-red (Fig. 1). Confocal images reveal a reticular pattern for R-CEPIAer and a more perinuclear and filamentous expression for G-CEPIA2mt (Fig. 1A), consistent with distribution of ER and mitochondria in HeLa cells25,26,27.

Figure 1
figure 1

Monitoring Ca2+ in the cytosol, ER and mitochondria simultaneously. (A) Expression pattern of the ER Ca2+ sensor R-CEPIAer and the mitochondria Ca2+ sensor G-CEPIA2mt expressed in HeLa cells. (B) Variations in Ca2+ levels in the cytosol (cyt), mitochondria (Mito), and ER during store depletion induced by histamine (His, grey shading) and thapsigargin in a Ca2+-free media (Tg, pink shading), and when SOCE is allowed by the re-addition of Ca2+ (2 mM, blue shading). Cytosolic Ca2+ was monitored using Fura-Red, the mitochondria using the fluorescence signal of G-CEPIA2mt, and the ER using R-CEPIAer. Left and right panels come from two different sets of experiments. (C) Bar chart summarizing the variations in Ca2+ levels in the cytosol and mitochondria during the protocols illustrated in A. (D) Ca2+ dynamics in response to the reversible SERCA blocker, cyclopiazonic acid (CPA). Cells were exposed to CPA (50 µM) for 10 min in Ca2+-free media, and then CPA was washed away for 10 min to allow the SERCA pumps to recover their function. Histamine was then applied (100 µM, 30 s) (pink shading), which failed to generate a large Cac2+ signal. Bringing back Ca2+ in the extracellular media (blue shading), replenishes ER stores and restores the responsiveness to histamine (grey shading). (E) Bar chart summarizing the variations in Ca2+ in the cytosol and in the mitochondria during the protocol illustrated in panel D. For reference, the Cac2+ and Cam2+ signal in response to SOCE induced with thapsigargin (panel C) are also illustrated (dotted lines). Values are given as means ± S.E.M, the number of cells analyzed are indicated on the charts. Statistics are calculated according to Student’s t-test, ANOVA and Tukey’s multiple comparisons test.

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Mobilizing Ca2+ stores with histamine (100 µM, 30 sec) results in a simultaneous rise in Cac2+ and Cam2+ showing that mitochondria readily detect Ca2+ released from stores when the stores are full. Ca2+ release was coupled to a transient depletion of ER stores which refill due to do recycling of released Ca2+ (Fig. 1B). However, ER refilling was incomplete due to the absence of extracellular Ca2+ and thus SOCE, resulting in a sustained low level depletion state of the ER (Fig. 1B), which could be reversed by adding Ca2+ to the media (not shown). The relative efficiency of the SERCA pump in capturing released Ca2+ in the absence of Ca2+ influx was variable from cell to cell. As indicated in Fig. 1B, the ER could partly recover from depletion without the contribution of SOCE. The recaptured amount of Ca2+ released in the absence of extracellular Ca2+ varied between 7% to 65% with a mean value of 30.1 ± 5.1% (given as a percentage of the reduction in ER signal and measured before extracellular Ca2+ re-addition) (Fig. 1B).

We then used the classical SOCE protocol of irreversible inhibition of SERCA with thapsigargin (TG, 1 µM) in Ca2+-free solution followed by Ca2+ addition to maximally activate SOCE and temporally isolate it. TG results in Ca2+ release coupled to emptying of ER Ca2+ stores but little to no increase in mitochondrial Ca2+ (Cam2+) (Fig. 1B). Replenishing extracellular Ca2+ results in cytosolic Ca2+ influx through SOCE that was not associated with store refilling since SERCA in blocked (Fig. 1B). It should be noted that the mitochondrial Ca2+ response was quite variable under this experimental paradigm, with only 56.5% of the cells showing a small mitochondrial Ca2+ transient in response to TG-dependent Ca2+ release; and 49.5% of cells responding to SOCE (n = 85 cells). The rise in Ca2+ recorded in the mitochondria was still significant in both cases (Fig. 1C). This confirms previous studies in HeLa cells showing limited coupling between the SOCE microdomain and the mitochondria23,24.

The peak amplitude of the global cytosolic Ca2+ signal was similar in response to Ca2+ release induced by histamine or thapsigargin, or following maximal SOCE induction (Fig. 1C). The mitochondria though responds in a dramatically distinct way to these similar cytosolic Ca2+ transients. Whereas mitochondria responded readily to the histamine induced Ca2+ rise, they responded poorly if at all to thapsigargin-induced Ca2+ release or SOCE (Fig. 1C). This argues for the existence of spatially defined Ca2+ microdomains, not detectable by current Ca2+ imaging approaches, but readily sensed by different effectors. Mitochondria could be taking up Ca2+ released from the ER directly through MAMs when the stores are full. To assess whether this is the case, we recorded at higher scanning speed (1 Hz) to determine the temporal relationship between Cac2+ and Cam2+. The rise in Cam2+ consistently followed the Cac2+ transient with a significant delay (3.4 ± 0.2 s, n = 54) (Supplemental Fig. 1), similar to what was reported previously28.

The SOCE signal recorded with thapsigargin is maximal and non-physiological, since thapsigargin irreversibly blocks SERCA and prevents store refilling. To obtain a better measure of physiological SOCE, we used the reversible SERCA inhibitor cyclopiazonic acid (CPA). CPA induced a store depletion and a Cac2+ rise similar to thapsigargin (61 ± 2% for Tg, n = 85 and 56 ± 3%, for CPA n = 29), without any significant increase in Cam2+ (Fig. 1D,E). The peak cytosolic Ca2+ release induced by CPA was slightly smaller than that induced by thapsigargin (0.64 ± 0.04 vs 0.80 ± 0.03, p < 0.05). Exposing the cell to histamine after CPA treatment in Ca2+-free solution resulted in a significantly reduced Ca2+ release (Fig. 1D,E, pink bar), showing that CPA efficiently depletes Ca2+ stores. When extracellular Ca2+ was reintroduced, it allowed quick refilling of the stores (Fig. 1D, blue bar), and a detectable but very small SOCE as a rise in Cac2+ (Fig. 1D,E, blue bar). The active refilling of the stores shows that CPA was washed out effectively and that SERCA is functional. The SOCE-dependent cytosolic Ca2+ transient detected under this experimental paradigm is orders of magnitude smaller than that detected with TG, yet it efficiently refills Ca2+ stores (Fig. 1D,E), arguing that SERCA is effective at limiting Ca2+ diffusion outside the SOCE microdomain into the bulk cytosol by sequestering Ca2+ flowing through SOCE channels into the ER lumen. Consistent with this interpretation, SOCE under these conditions does not induce any mitochondrial Ca2+ rise (Fig. 1D,E). When histamine was applied after reloading of the ER stores, it elicited a large rise in Cac2+ and Cam2+ indicating normal function of the Ca2+ signaling machinery and no deleterious effects of the experimental protocol (Fig. 1D,E, grey bar).

Mitochondria do not respond to Ca2+ tunneling

The above data show that the mitochondria in HeLa cells do not respond to Ca2+ entry through SOCE. However, the experimental conditions above using CPA followed by a wash do not result in the opening of IP3 receptor and as such would not allow for Ca2+ tunneling as would be expected in response to agonist stimulation. To directly test whether the mitochondria act as a downstream effector of Ca2+ tunneling, we devised a protocol to temporally isolate Ca2+ tunneling from the Ca2+ release phase. We depleted Ca2+ stores with CPA (10 min) followed by a wash to release SERCA from inhibition in Ca2+-free conditions (Fig. 2A). This was followed by the addition of histamine (100 µM, 30 s) concurrently with extracellular Ca2+ re-addition, thus opening IP3 receptor and allowing for Ca2+ tunneling (Fig. 2A). Surprisingly, histamine and Ca2+ application extracellularly under these conditions results in a large cytosol Ca2+ rise of similar amplitude to that induce by histamine when Ca2+ stores are full (Fig. 2A,B). Under these conditions, histamine cannot induce Ca2+ release since the stores are empty due to the CPA treatment (see Fig. 1D, pink bar). Therefore, the large Ca2+ transient observed under these conditions requires that Ca2+ entering the cell through SOCE be first pumped into the ER by SERCA and then released through IP3R, i.e. the classical tunneling pathway. To confirm that our experimental approach induces Ca2+ tunneling, we analyzed the time course of the Cac2+ rise due to tunneling as compared to ER refilling. As shown in Fig. 2C, the Cac2+ signal due to Ca2+ tunneling precedes the initiation of ER refilling by tens of seconds, a phase during which Ca2+ flowing through SOCE is preferentially taken up into ER stores and release again through IP3R to expand the spatial spread of SOCE. Presumably, the large conductance of IP3Rs prevents store refilling during this phase. As IP3 is metabolized IP3Rs close allowing SERCA to refill the stores (Fig. 2C). To confirm our interpretation that during Ca2+ tunneling Ca2+ leak through IP3 receptors delays store refilling, we superimposed the time course of ER refilling during Ca2+ tunneling as in Fig. 2A (blue bar) with that during Ca2+ refilling in the absence of histamine as in Fig. 1D (blue bar). There is a clear statistically significant (p < 0.001) delay in store refilling during tunneling where the stores require 106.4 + 7.0 sec to reach half-maximal filling compared to 62.1 + 5.3 sec in the absence of histamine (Fig. 2D,E).

Figure 2
figure 2

Mitochondria do not respond to Ca2+ tunneling. (A) To temporally isolate Ca2+ tunneling, Ca2+ stores were depleted using CPA, followed by a wash out of CPA, and then histamine (100 µM, 30 s) was applied together with extracellular Ca2+ (blue shading). This induces Ca2+ entry through SOCE, with SERCA active and open IP3Rs to allow for Ca2+ tunneling. When the ER stores have regained their original level, histamine is applied again (grey shading). (B) Bar chart summarizing the variations in Ca2+ levels in the cytosol and mitochondria during the protocols illustrated in panel A. (C) Average time course of the cytosolic Ca2+ signal (purple) and of the ER Ca2+ signal (red) during Ca2+ tunneling. (D) Comparative time course of the refill kinetics of the ER during Ca2+ tunneling (red) as compared to during physiological SOCE (green, as in Fig. 1D blue bar). (E) Quantification of the refill time (measured at 50% of the maximum refill) during physiological SOCE and during tunneling. Data are means ± S.E.M, statistics are performed using a paired Student’s t-test, the number of cells is indicated in each panel.

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Surprisingly though and despite its large amplitude, the Cac2+ rise due to tunneling did not produce a Ca2+ rise in the mitochondria (Fig. 2A,B; blue bar). However, when the stores where replenished, a second identical application of histamine resulted in a large rise in Cam2+ (Fig. 2A,B, grey bar). This shows that Ca2+ signals generated by Ca2+ tunneling downstream of SOCE or by agonist-dependent mobilization when Ca2+ stores are full are not equivalent in terms of inducing a Ca2+ response in the mitochondria despite the fact that they result in an equivalent Cac2+ rise and are both mediated through IP3 receptors. Therefore, Ca2+ tunneling and Ca2+ release are distinct in their ability to activate downstream effectors. These results argue that the coupling between Ca2+ influx through SOCE, uptake by SERCA and release into the cytosol when Ca2+ tunneling is operational is highly efficient at raising cytosolic Ca2+ in a spatially localized fashion as it is not detected by mitochondria.

We further confirmed that Ca2+ flowing through SOCE is not limiting in terms of producing a Cam2+ response in these experiments by raising extracellular Ca2+ to 10 mM during the tunneling protocol to increase Ca2+ flow through SOCE. This results in a similar cytoplasmic signal and did not restore the mitochondrial signal (Supplemental Fig. 2). Another prediction from the Ca2+ tunneling pathway is that it would be slower in mediating the Cac2+ rise as compared to Ca2+ release on full stores. The tunnel mechanism requires two more steps as compared with Ca2+ release: first Ca2+ influx through SOCE channels and second uptake in the ER lumen by SERCA, independently of a potential delay required for the diffusion of Ca2+ into the ER cisternae. It was not possible to resolve this potential delay in the imaging experiments of the three compartments simultaneously because of the slow sampling speed (0.1 Hz). To allow a higher speed of recording (1 Hz), we loaded the cells solely with the Ca2+ indicator Fluo4-AM and performed the same protocol as in Fig. 2. The analysis of the time course using either a “virtual” line scan or a global measurement of the Cac2+ over time shows a significantly slower rise in the Cac2+ mediated by Ca2+ tunneling as compared to Ca2+ release on full stores, although both signals ultimately reached similar amplitudes (Supplemental Fig. 3).

Another formal possibility in mediating the mitochondrial Ca2+ respond to Ca2+ release as compared to Ca2+ tunneling is the spatial localization of the mitochondria in relationship to the Ca2+ source. Depending on the cell type (and physiological conditions) the mitochondria can be positioned close to the plasma membrane as in immune cells where they regulate SOCE19,20 or deeper in the cell where they preferentially interact with the ER23. We therefore tested whether store depletion affects the relative position of mitochondria in HeLa cells. However, we could not detect changes in mitochondrial localization using either confocal or TIRF imaging (Supplemental Fig. 4). We also evaluated the changes in the morphology of the mitochondria after store depletion using 3D imaging, although we detected some reduction (9%) in the length of mitochondria branches the effect was small and irreversible after Ca2+ readdition, and therefore unlikely to explain the differential effect of Ca2+ and Ca2+ tunneling on mitochondria (Supplemental Fig. 5).

Taken together our results reveal that given the spatio-temporal dynamics of Ca2+ release versus Ca2+ tunneling, and despite the fact that both rely on IP3Rs and are associated with an equivalent rise in global Cac2+, mitochondria respond readily to Ca2+ release but not Ca2+ tunneling. We hypothesize that the slower speed of Cac2+ rise during tunneling, combined with the distance between the point source of Ca2+ entry (the Orai channel) and the target (i.e. the mitochondria) impair the formation of a “Hot Spot” or high Ca2+ domain between the ER and the mitochondria that would allow the MCU to import Ca2+ during tunneling.

Ca2+-activated potassium channels

The global Ca2+ rise detected following tunneling argues for a spatially localized cytosolic Ca2+ transient. The mitochondria, which tend to localized deeper within HeLa cells, are unable to detect this transient. In Xenopus oocytes Ca2+ tunneling is particularly effective at stimulating Ca2+-activated Cl channels at the PM7. Therefore, to test whether Ca2+-dependent PM localized effectors respond to Ca2+  tunneling, we turned to Ca2+-activated K+ channels (KCa), which are expressed in HeLa cells29. Histamine-dependent Ca2+ release when stores are full induces a transient KCa current (Fig. 3A and Supplemental Fig. 6). Similarly, Ca2+ tunneling was effective at activating KCa (Fig. 3A,B, Tun), but with a smaller peak amplitude than the current induced by histamine from full stores (Fig. 3A,B, Rel). The tunneling-induce KCa was longer lasting though, leading to a larger charge transfer for an identical histamine stimulation (Fig. 3B). Comparatively, a much smaller maximal gKCa current was observed in response to SOCE, using the CPA-wash protocol (Fig. 3B, SOCE). This indicates that Ca2+ tunneling activates gKCa at the PM, significantly more efficiently than SOCE (Fig. 3B), in a similar fashion to what is observed with Ca2+-activated Cl channels7. This is consistent with data from human submandibular gland cells where gKCa activation by Ca2+ influx required the uptake of Ca2+ in the ER stores30.

Figure 3
figure 3

Ca2+-activated K+ channels in response to Ca2+ tunneling and Ca2+ release. (A) Cells were voltage-clamped in the whole-cell configuration at 0 mV to enhance the driving force for K+. The bath application of histamine (100 µM, 30 s) in a Ca2+-free media induced a transient outward current. When Ca2+ tunneling is induced a slow developing outward current is observed. (B) Bar charts summarizing the current amplitude and charge transfer (summed over a 5 min period) obtained in response to SOCE (after CPA treatment), Ca2+ tunneling (Tun), Ca2+ release on full stores (Rel). Statistics are according to Student’s unpaired t-test.

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The differential response of gKCa and the mitochondria argue that Ca2+ tunneling is effective at raising cytosolic Ca2+ levels in the cell cortex close to the PM but not deep within the cell. To record gKCa we used the whole-cell patch-clamp, which modifies the intracellular environment after breaking into the cell and could have deleterious effects on Ca2+ buffering and spatial dynamics. We therefore sought a non-invasive approach to visualize cortical Ca2+ transients and confirm the results obtained with gKCa. We used a membrane targeted Ca2+-sensor Lck-GCamp5G31, coupled to TIRF microscopy to measure Ca2+ changes specifically at plasma membrane level. Bath application of histamine induced a fast transient elevation of Ca2+ in the sub-PM layer (Fig. 4A,B). Comparatively, Ca2+ influx through ‘physiological’ SOCE activated using the CPA-washout protocol results in a smaller, slower and longer lasting cortical Ca2+ increase (Fig. 4B). In contrast, when SOCE was induced maximally with thapsigargin it results in larger cortical Ca2+ influx (Fig. 4B,C), thus confirming that irreversible inhibition of SERCA enhances cortical Ca2+ transients. In agreement with the gKCa data, Ca2+ tunneling results in a slow and long lasting increase in sub-PM Ca2+ of significantly higher amplitude and duration then SOCE (Fig. 4B,C). In a pattern similar to what we observed with gKCa the amplitude of the tunneling signal was smaller than the release induced by histamine on full stores, but significantly longer lasting, creating a larger transfer of total Ca2+ (evaluated using the area under the Ca2+ traces) (Fig. 4D). Interestingly, during tunneling the total amount of Ca2+ ions in the cell cortex is equivalent to that observed with thapsigargin-induced SOCE (Fig. 4D). This highlights the efficiency of the pump-leak pathway at the ER membrane during tunneling, and the conversion of the slow conductance of SOCE channels into a sustained signal at the sub-PM using the high-conductance leak of IP3 receptors.

Figure 4
figure 4

TIRF imaging of Ca2+ dynamics in the cell cortex using Lck-GCamp5G. (A) Representative TIRF images of the fluorescence of the plasma membrane-anchored Ca2+ sensor Lck-GCamp5G at rest (Ctr) and during application of histamine (His; 100 µM, 30 s). (B) Typical examples of the time course of the different Ca2+ signals recorded under the plasma membrane following: Ca2+ release from full Ca2+ stores with Histamine (Release); Ca2+ tunneling with SOCE re-fueling the ER and releasing Ca2+ through IP3Rs stimulated by histamine (Tunnel); and SOCE induced by CPA after washout or after store depletion by thapsigargin (Tg). (C) Bar chart summarizing the peak amplitude of the SOCE signals induced after store depletion with either thapsigargin or CPA, and that induced following Ca2+ release and in response to Ca2+ tunneling. (D) To account for the difference in the signal kinetics, the area under the trace was integrated over a 5 min period and summarized in a bar char. The number of cells is indicated above the bars, statistics are according to ANOVA followed by Tukey’s multiple comparison test.

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NFAT1 translocation

A well characterized effector that responds exquisitely to Ca2+ in the SOCE microdomain and not Ca2+ release from stores is the calcineurin-NFAT signaling pathway14,32. NFAT1 is a transcription factor, phosphorylated at rest and dephosphorylated following the activation of calcineurin by SOCE, which leads to its translocation to the nucleus (Fig. 5A). The effect of Ca2+ tunneling on gKCa argues that it extends the SOCE Ca2+ microdomain in the cortical region of the cell and activators effectors accordingly. Therefore, Ca2+ tunneling should not affect the activation of calcineurin-NFAT as it is not expected to alter the SOCE microdomain. We therefore tested NFAT nuclear translocation in response to Ca2+ release, SOCE and Ca2+ tunneling (Fig. 5). Consistent with previous reports, Ca2+ release induced by thapsigargin, CPA or histamine did not induce NFAT1 translocation (Fig. 5B). In contrast, when SOCE was activated with either thapsigargin or CPA, it effectively induces NFAT1 translocation to the nucleus although it occurred with a faster time course in response to TG (Fig. 5A–C). Ca2+ tunneling results in higher levels of NFAT1 translocation with similar kinetics as those observed in response to CPA (Fig. 5C). This is likely due to the longer duration of the Ca2+ signal in the SOCE microdomain when Ca2+ tunneling is active, due to the continuous pump-leak of Ca2+ at the ER membrane. Alternatively, it may indicate some additional activation of calcineurin outside the SOCE microdomain in the sub-PM domain. Therefore, as expected Ca2+ tunneling does not dramatically alter the activation of the calcineurin-NFAT axis.

Figure 5
figure 5

NFAT1 translocation induced by different Ca2+ mobilizing mechanisms. (A) Wide-field fluorescence images of the translocation of NFAT1 from the cytoplasm to the nucleus after store depletion with thapsigargin and activation of SOCE. (B) Maximum change in the nucleo-cytoplasmic ratio of NFAT1 in response to Ca2+ release from stores (Ca Release) using thapsigargin (Tg), CPA or histamine; or in response to Ca2+ influx through SOCE induced by Tg or CPA or in response to Ca2+ tunneling (Tun). (C) Example time courses of the translocation of NFAT1 induced by SOCE or by Ca2+ tunneling. The time constant was measured in all three conditions and summarized in a bar chart (inset). The number of experiments is indicated above or inside the bars, statistics are according to ANOVA followed by Tukey’s multiple comparison test.

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Discussion

Agonist stimulation through GPCRs or receptor tyrosine kinases often couples to PLCs resulting in the generation of Ca2+ transients that in non-excitable cells tend to be biphasic, with the initial release phase due to Ca2+ mobilization from intracellular Ca2+ stores being rapid and of high amplitude but short lived as the stores empty. This is followed by a sustained phase of Ca2+ influx, with varying duration based on the cell type and the agonist, that is due to the activation of SOCE. Experimentally, protocols have been devised to temporally separate the Ca2+ release and SOCE phases as it allows for a better dissection of their relative contributions. However, physiologically the two processes are tightly linked and overlapping with SOCE being activated as stores gradually deplete and most likely in a spatially complex fashion. Therefore, in the cycle of IP3-dependent Ca2+ release, store depletion, SOCE activation, store refilling and SOCE inactivation there is a time window where SOCE is active while IP3 receptors are still open resulting in a pump-leak at the ER membrane, due to Ca2+ uptake by SERCA within the SOCE microdomain while Ca2+ is released through open IP3 receptors, a process known as Ca2+ tunneling. Conceptually this is reminiscent of the ‘capacitative Ca2+ influx’ model originally proposed by Jim Putney33, where he envisioned Ca2+ flowing directly from the extracellular space into the ER lumen to refill the stores, before the plethora of signaling roles of SOCE in addition to store refilling were appreciated. Ca2+ tunneling adds a new dimension for Ca2+ signaling downstream of SOCE by allowing Ca2+ influx to activate effectors that are spatially far away from the point source entry at SOCE puncta. This would be essential for SOCE to activate different effectors that do not localize to the SOCE microdomain, especially in cells such as HeLa where the SOCE puncta after store depletion are estimated to occupy <1% of the PM12. Furthermore, an essential feature of Ca2+ tunneling is to bypass the highly buffered cytosol to allow Ca2+ ions to reach their target by using the ER lumen as a tunnel given it’s lower buffering capacity34.

Ca2+ tunneling was originally described in pancreatic acinar cells where it transports Ca2+ entering at the basolateral side of the cell through ER tunnels to the apical side where IP3Rs localize (see Petersen et al. 2017 for a recent review)2,16. Ca2+ tunneling was more recently generalized through studies in frog oocytes, showing dramatic remodeling of the Ca2+ signaling machinery at the PM in response to store depletion to support Ca2+ tunneling, where it targets both Ca2+-activated Cl channels and the IP3R itself to modulate tonic versus oscillatory Ca2+ signaling7,35. The same mechanism of using ER tunnels to transport Ca2+ to effectors has been alluded to, although not directly investigated, in other studies where it targets the Ca2+-activated K channel, nuclear NFAT activation, and Ca2+ transport from the soma to maintain Ca2+ signaling in dendrites14,30,36.

In this study, we were interested in testing the functionality of Ca2+ tunneling in non-polarized or specialized cells that differ from pancreatic acinar cells, neurons or oocytes for instance, using multiple different effectors of different nature with distinct spatial distribution (organelle, channel, and signaling molecule). We use simultaneous imaging of the three primary Ca2+ signaling compartments in HeLa cells (cytosol, ER and mitochondria) to assess the functionality of the Ca2+ tunneling mechanism and its ability to specifically and selectively activate downstream effectors. Our results show that Ca2+ tunneling is functional in HeLa cells downstream of store depletion (Fig. 2), where using combinations of agonist stimulation, manipulation of extracellular Ca2+, and a simple CPA-wash protocol, allows us to separate Ca2+ release, SOCE and Ca2+ tunneling to assess the effect of each Ca2+ signaling modules on downstream efforts. At the outset of the study an attractive target for Ca2+ tunneling was the mitochondria given the well documented intimate physical interaction between the ER and mitochondria through the MAMs, and their distribution away from the PM in HeLa cells23. Surprisingly, we show that mitochondria in HeLa cells respond readily to agonist-dependent Ca2+ release when stores are full with a delay after a Cac2+ rise (Fig. 1), but do not respond to Ca2+ tunneling despite the fact that the Cac2+ signal reaches a similar amplitude globally (Fig. 2). In contrast Ca2+ tunneling was more effective than Ca2+ release and far more effective than SOCE at activating gKCa and at raising sub-PM Ca2+ levels. Mitochondria in HeLa cells do not respond well to Ca2+ flowing through SOCE23, a finding that we have confirmed here (Fig. 1). However, the mitochondrial response to Ca2+ tunneling is even poorer than to maximal SOCE stimulated by thapsigargin (Fig. 2B). The chain of channels and pumps mediating Ca2+ tunneling in series could potentially explain this observation. Ca2+ influx through SOCE initiates Ca2+ tunneling followed by Ca2+ uptake into the depleted ER through SERCA activity, and then release through open IP3Rs. The limiting factor as discussed below in this Ca2+ transport chain is the SERCA pump given its low flux. Estimates of the single channel Ca2+ current through the IP3R under physiological conditions are 0.1-0.2 pA37, corresponding to a rate of 6 × 105 Ca2+/sec at the lower end of the spectrum. The rate of SERCA2b uptake was estimated at ~40 Ca2+/sec at Vmax6,38, and the flux through an Orai channel is estimated at 5000 Ca2+/sec considering a Po of 0.8 (see Hogan for a detailed discussion)6. These estimates argue that during Ca2+ tunneling the leak through any open IP3R is orders of magnitude higher than the trickle of Ca2+ that can be fed into the ER by SERCA. This would ensure that the ER does not refill readily allowing Ca2+ tunneling to proceed for tens of seconds. Assuming that IP3Rs are evenly distributed throughout the ER membrane and opening stochastically the first open IP3R encountered would release the Ca2+ fed into the ER by 15,000 SERCA pumps operating at full capacity upstream. This implies that during tunneling Ca2+ entering the cell through SOCE would not be able to travel deep within the cell as the ER acts as a sieve with open IP3Rs resulting in Ca2+ leak from the cortical ER, thus preventing Ca2+ from reaching deep within the ER to activate mitochondria (Fig. 6). This model is attractive, as it would also explain the differential response of gKca and mitochondria to Ca2+ tunneling (Fig. 6). The remodeling of the Ca2+ signaling machinery in response to store depletion results in SOCE as a point source Ca2+ entry that localizes to focal sites at the PM and as such directionally feeding Ca2+ into the cell. At this point, we cannot rule out other contributing factors such as the slower speed of Ca2+ tunneling as compared to Ca2+ release or some kind of restructuring to the MAMs (Supplemental Fig. 3).

Figure 6
figure 6

Cartoon model of the spatial distribution of Ca2+ signals at the SOCE microdomain, ER lumen, cytosol and mitochondria during SOCE, Ca2+ release on full stores, and Ca2+ tunneling. See text for details.

Full size image

The flux estimates discussed above would also predict leakage of Ca2+ from the SOCE microdomain since the flow of Ca2+ through Orai channels in SOCE puncta would be predicted to overwhelm SERCA pumps that localize to the puncta. Consistently, we observe an increase in sub-PM Ca2+ and activation of gKca in response to SOCE. However, Ca2+ tunneling greatly enhance both as would be expected from the leak through IP3Rs (Fig. 6). Furthermore, Ca2+ tunneling by maintaining the ER depleted extends the duration of SOCE and allows it to more effectively activate downstream effectors. As shown in Fig. 3, although the amplitude of gKCa induced by Ca2+ tunneling is much smaller than that induced by Ca2+ release, the total charge transfer is significantly greater in response to Ca2+ tunneling. This is reflected as well in the Ca2+ signal in the sub-PM domain (Fig. 4).

NFAT activation in contrast to gKCa or the mitochondrial response, is quite specific to a Ca2+ rise in the SOCE microdomain as it is activated with equal efficiency whether SOCE is induced maximally using thapsigargin or to physiological levels using the CPA-wash protocol (Fig. 5). This is consistent with previous studies14,32,39. There is a statistically significant increase in NFAT translocation when Ca2+ tunneling is activated (Fig. 5B). This could be due to either the increased duration of the Ca2+ signal in the SOCE microdomain due to Ca2+ tunneling (Fig. 4B) or alternatively activation of calcineurin that localizes outside the SOCE clusters that can be targeted by Ca2+ tunneling.

Collectively, our data show that Ca2+ tunneling is functional in HeLa cells and expands the specificity of the Ca2+ signaling machinery toward downstream effectors and sub-cellular domains. The response of the mitochondria and gKCa shows that Ca2+ tunneling is particularly effective at raising Ca2+ levels in the cortical cytoplasm next to the PM. This can be explained by the high conductance of IP3Rs as discussed above, leading to a strong leak that favors Ca2+ release in the cortical region close to the SOCE point source Ca2+ entry. It therefore appears that Ca2+ tunneling is a specialized module to raise cortical Ca2+ levels thus effectively expanding the SOCE microdomain. This is somewhat distinct from the long range Ca2+ transport due to tunneling in pancreatic acinar cells, which supports the transport of Ca2+ through the ER lumen from the basolateral to the luminal end of the cell. Ca2+ tunneling in addition to expanding the spatial spread of the SOCE microdomain also modulates the temporal dynamics of SOCE by extending the duration of SOCE by maintaining a depleted ER and lower Ca2+ levels in the SOCE microdomain due to the continuous pumping of Ca2+ into the ER lumen by SERCA and its release through IP3Rs (Fig. 6). Finally, and consistent with our findings, Thillaiappan et al. recently showed that licensed IP3Rs preferentially localize close to the PM in the cell cortex and at the periphery of STIM1 clusters40. Such a localization of immobile IP3Rs is ideally suited to support Ca2+ tunneling and in particular providing it with its cortical spatial specificity as discussed herein.

Methods

Cell culture and solutions

Hela cells were cultured in DMEM media containing 10% fetal bovine serum supplemented with penicillin (100 units.ml−1) and streptomycin (100 µg.ml−1). The cells were plated 24 h before transfection on poly-lysine coated glass-bottom dishes (MatTek, U.S.A). For all live cells experiments, the cells were continuously perfused using a peristaltic pump (Gilson Minipuls) at the speed of 1 ml.min−1. The standard saline contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES, pH 7.2, for Ca2+-free experiments, the Ca2+ was exchanged equimolarly with Mg2+.

Intracellular Ca2+ imaging

To perform organelle Ca2+ imaging HeLa cells were transfected with the Ca2+ indicators G-CEPIA2mt and R-CEPIAer constructs (0.5 µg per dish) using a standard lipofectamine 2000 (Invitrogen) procedure 24 h to 48 h prior to imaging. Both constructs were obtained from addgene (#58218 and #58216 respectively) and were originally created by Masamitsu Lino’s group25. To image cytoplasmic Ca2+ the cells were loaded for 45 min with Fura Red-AM at 37 °C (1 µM from a 1 mM stock in 20% pluronic acid/DMSO). Imaging was performed on a Leica TCS SP5 confocal system (Leica, Germany) fitted with a 63x/1.4-06 oil immersion objective using an open pinhole. The G-CEPIA2mt was excited using a 488 nm laser line and the emission collected at 500–590 nm. The same line was used to excite the Ca2+-free form of FuraRed and the emission collected between 600–709 nm. For R-CEPIAer the excitation was performed with a 561 nm laser line and the signal collected between 583–649 nm. The frame rate was set to 0.1 Hz unless stated otherwise. For Fluo4 imaging, the cells were loaded with 1–2 µM Fluo4AM (45 min/37 °C). The excitation was performed using a 488 nm laser line and the signal collected at 500–560 nm with the pinhole at 1 airy unit and the frame rate was set to 1 Hz.

Morphological analysis

Cells expressing G-CEPIA2mt and R-CEPIAer were fixed (PFA 4%, 10 min) and stained with Alexa633 tagged Wheat Germ Agglutinin (2 µg.ml−1) (Invitrogen). Confocal images were acquired every 0.5 µm to generate z-stacks. The imaging was performed on a Zeiss LSM880 controlled by Zen Black 2.3 (Zeiss, Germany) and fitted with a 63x/1.4 objective. The imaging parameters were as follows: for G-CEPIA2mt: λex = 488 nm, λem = 494–568 nm, for R-CEPIAer: λex = 561 nm, λem = 570–622 nm and for WGA-Ax633: λex = 633 nm, λem = 640–747 nm. The distance between the plasma membrane, the ER and the mitochondria was evaluated using the profile of a linear region of interest drawn between both sides of the cell and the distance measured at 50% of the peak amplitudes of the signals.

Whole-cell patch-clamp

The Ca2+ activated K+ channels were recorded using a standard whole-cell patch-clamp protocol. Patch pipettes (resistance ranging from 4 to 6 MΩ when filled with the pipette solution) were sealed to the plasma membrane and the patch ruptured after the formation of a giga-ohm seal. The cells were voltage-clamped at 0 mV at steady state using an Axopatch 200B amplifier (Molecular Devices, U.S.A) controlled by pClamp 10. The internal pipette solution contained (in mM) 140 K-Gluconate, 2 NaATP, 2 MgCl2, 10 HEPES, 1 µM EGTA and pH 7.4. The extracellular solutions and perfusion system was the same as the imaging experiments.

Total Internal Reflection Fluorescence Microscopy (TIRF)

For the localization at the plasma membrane, the cells where transfected with the Orai1-mCh, STIM1-CFP and CEPIA2mt constructs, and the stores depleted with thapsigargin. The membrane plane was identified by the presence of clusters of STIM1 and Orai1, and used to adjust the evanescent wave. The imaging was performed on a Zeiss Cell observer TIRF system using the following parameters for STIM1-CFP: λex = 405 nm, λem = 446–468 nm, for G-CEPIA2mt: λex = 488 nm, λem = 510–555 nm and for Orai1-mCh: λex = 561, λem = 581–679 nm. For TIRF Ca2+ imaging at the plasma membrane, the cells were transfected with the Ca2+ sensor Lck-GCamp5G (Addgene #34924)31. The sensor was excited at λex = 488 nm and the images collected using λem = 510–555 nm, the frame rate was 0.1 Hz. The perfusion system and solutions was identical to the previous experiments.

NFAT1 translocation

Cells were transfected for 24 h with the NFAT1-GFP construct (Addgene #11107)41, the imaging was performed using the same settings as the TIRF imaging for GFP-tagged proteins except that the mirror was set vertically to obtain a widefield image. The perfusion saline and system were as previously described.

Data analysis and statistics

The imaging data was quantified using FIJI/ImageJ 1.51 n42,43 and ZenBlue 2.3 (Zeiss). The patch-clamp data was analyzed with Clampfit 10.0 (Molecular Devices). Statistics and data analysis were performed using Graphpad Prism 7.02 (GraphPad U.S.A). Values are given as means ± S.E.M and statistics were performed using either paired or unpaired Student’s t-test or ANOVA followed by Tukey’s test for multiple comparisons. P-values are ranked as follows *P < 0.05, **P < 0.01, ***P < 0.001.