Spatially restricted subcellular Ca2+ signaling downstream of store-operated calcium entry encoded by a cortical tunneling mechanism

Abstract

Agonist-dependent Ca²⁺ mobilization results in Ca²⁺ store depletion and Store-Operated Calcium Entry (SOCE), which is spatially restricted to microdomains defined by cortical ER - plasma membrane contact sites (MCS). However, some Ca²⁺-dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca²⁺ tunneling, mediates the uptake of Ca²⁺ flowing through SOCE into the ER followed by release at distal sites through IP₃ receptors. Here we show that Ca²⁺ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca²⁺ release when stores are full, SOCE shows little effect in raising mitochondrial Ca²⁺, and Ca²⁺-tunneling is completely inefficient. In contrast, gK_Ca displays a similar sensitivity to Ca²⁺ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca²⁺ release. These results show that in contrast to the previously described long-range Ca²⁺ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca²⁺ rise within the cortical region of the cell to activate a specific subset of effectors.

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 Ca_c²⁺ 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 Ca_c²⁺ and Ca_m²⁺ 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.

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.

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.

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.

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.

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.