Ca²⁺ shuttling between endoplasmic reticulum and mitochondria underlying Ca²⁺ oscillations

Abstract

Although many cell functions are regulated by Ca²⁺ oscillations induced by a cyclic release of Ca²⁺ from intracellular Ca²⁺ stores, the pacemaker mechanism of Ca²⁺ oscillations remains to be explained. Using green fluorescent protein-based Ca²⁺ indicators that are targeted to intracellular Ca²⁺ stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca²⁺ shuttles between the ER and mitochondria in phase with Ca²⁺ oscillations. Following agonist stimulation, Ca²⁺ release from the ER generated the first Ca²⁺ oscillation and loaded mitochondria with Ca²⁺. Before the second Ca²⁺ oscillation, Ca²⁺ release from the mitochondria by means of the Na⁺/Ca²⁺ exchanger caused a gradual increase in cytoplasmic Ca²⁺ concentration, inducing a regenerative ER Ca²⁺ release, which generated the peak of Ca²⁺ oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca²⁺ was depleted. Thus, Ca²⁺ shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca²⁺ oscillations.

Introduction

Ca²⁺ oscillation is a periodic spiking of intracellular Ca²⁺ concentration observed in many types of cell under stimulation (Clapham, 1995; Berridge et al, 2000), and Ca²⁺-dependent molecules are often regulated by the frequency of Ca²⁺ oscillations (De Koninck & Schulman, 1998; Dolmetsch et al, 1998; Li et al, 1998; Oancea & Meyer, 1998; Tomida et al, 2003). Although the importance of Ca²⁺ oscillations is widely recognized, the mechanism underlying Ca²⁺ oscillations remains to be explained. In many cell types, Ca²⁺ release by means of the inositol 1,4,5-trisphosphate receptor (IP₃R) has a pivotal role in the generation of Ca²⁺ oscillations. The IP₃R function is regulated not only by IP₃ concentration, but also by cytoplasmic Ca²⁺ concentration (Iino, 1990; Bezprozvanny et al, 1991). The Ca²⁺-mediated regulation of IP₃R is essential for Ca²⁺ oscillations, because cells expressing a mutant IP₃R with a reduced sensitivity to Ca²⁺ do not generate Ca²⁺ oscillations (Miyakawa et al, 2001). Although the endoplasmic reticulum (ER), which carries IP₃R, is mainly responsible for the generation of Ca²⁺ oscillations, mitochondria may also have a role as a buffer of cytoplasmic Ca²⁺ concentration (Jouaville et al, 1995; Rizzuto et al, 1998). However, the functions of these organelles in the generation of Ca²⁺ oscillations require further clarification.

We measured Ca²⁺ concentration dynamics within intracellular Ca²⁺ stores simultaneously with cytoplasmic Ca²⁺ concentration using green fluorescent protein (GFP)-based Ca²⁺ indicators that were designed to monitor Ca²⁺ concentration within either the ER or mitochondria. Our results show that Ca²⁺ concentrations in both ER and mitochondria oscillate concomitantly with cytoplasmic Ca²⁺ oscillations. There are, however, important phase differences in oscillatory change among the three compartments. Our results indicate that Ca²⁺ shuttling between the ER and mitochondria is an essential component of the Ca²⁺ oscillation mechanism, and has a pacemaker role in Ca²⁺ oscillations.

Results and Discussion

Ca²⁺ measurement within ER

To monitor Ca²⁺ concentration inside the ER ([Ca²⁺]_er), we used the GFP-based Ca²⁺ indicator (cameleon) after modifications (split-YC7.3er, K′_d≈130 μM; see supplementary information and supplementary Fig 1 online). We examined whether the indicator detects [Ca²⁺]_er in living cells. HeLa cells were sequentially transduced with retroviruses that encode the two parts of the indicator, and both parts were expressed in the cytoplasmic meshwork structure of the ER (supplementary Fig 2 online). On application of histamine, F480 increased, with a concomitant decrease in F535, as expected from a decrease in [Ca²⁺]_er (Fig 1A, upper diagram). We used F₅₃₅/F₄₈₀ as an indicator of [Ca²⁺]_er (Fig 1A, lower diagram). After histamine application, F₅₃₅/F₄₈₀ decreased without any noticeable delay after the increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt; Fig 2A,C,E,F), indicating that the indicator is not saturated with a luminal Ca²⁺ concentration in the resting state. When ER Ca²⁺ was subsequently depleted by inhibiting sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) by thapsigargin, F₅₃₅/F₄₈₀ decreased below the trough of histamine-induced oscillatory changes (Fig 1B). These results indicate that the dynamic range of split-YC7.3er covers the entire range of changes in [Ca²⁺]_er during histamine-induced Ca²⁺ oscillations. We did not observe any distinct spatial inhomogeneity in [Ca²⁺]_er measurements within cells at the present time resolution (Fig 1C).

Figure 1.

Measurement of [Ca²⁺]_er using split-YC7.3er. (A) Fluorescence change in a cell expressing split-YC7.3er cameleon after histamine stimulation. Fluorescence intensities (434 nm excitation) at 535 (F₅₃₅) and 480 nm (F₄₈₀; upper diagram) and their ratio F₅₃₅/F₄₈₀ (lower diagram). (B) Time course of F₅₃₅/F₄₈₀ during application of histamine and thapsigargin. Data are representative of five experiments. (C) Time courses of F₅₃₅/F₄₈₀ at different areas within the cell shown in the left panel. Data are representative of four experiments.

Figure 2.

Phase difference between [Ca²⁺]_er and [Ca²⁺]_cyt. (A) Simultaneous measurement of [Ca²⁺]_er and [Ca²⁺]_cyt using split-YC7.3er cameleon and indo-5F, respectively, during histamine application in the presence of extracellular Ca²⁺ (2 mM). Data are representative of ten experiments. (B) Expanded time courses of [Ca²⁺]_er and [Ca²⁺]_cyt of second Ca²⁺ oscillation in (A). (C) [Ca²⁺]_er and [Ca²⁺]_cyt in the absence of extracellular Ca²⁺ (2 mM EGTA). Data are representative of 13 experiments. (D) Second Ca²⁺ oscillation in (C) shown on expanded time scale. (E) Initial Ca²⁺ oscillation in (C) shown on expanded time scale. (F,G) Averaged and normalized time courses of the first Ca²⁺ oscillations (F), and the second and third Ca²⁺ oscillations (G) in the absence of extracellular Ca²⁺. Data are mean±s.e.m. (n=13).

[Ca²⁺]_cyt versus [Ca²⁺]_er

We then carried out simultaneous measurements of [Ca²⁺]_cyt and [Ca²⁺]_er using indo-5F and split-YC7.3er, respectively. As shown in Fig 2A, histamine induced oscillatory changes in [Ca²⁺]_cyt and [Ca²⁺]_er in opposite directions. Ca²⁺ oscillations were also observed in the absence of the extracellular Ca²⁺ (Fig 2C). The following analyses mainly use data in the absence of extracellular Ca²⁺, because the absence of Ca²⁺ influx through the plasma membrane simplifies the analyses.

Fig 2E shows the very first Ca²⁺ oscillation in response to histamine application. [Ca²⁺]_cyt increased rapidly, reaching a peak in ∼4 s. Conversely, the [Ca²⁺]_er change had the same onset time as the [Ca²⁺]_cyt change, but [Ca²⁺]_er continued to decrease after [Ca²⁺]_cyt reached its peak. The time courses of [Ca²⁺]_er and [Ca²⁺]_cyt during the first oscillations in different experiments were averaged after normalization (Fig 2F; see the supplementary information online for normalization procedure). The main features of the typical data are discernible in the averaged data. Thus, at the peak [Ca²⁺]_cyt, only 41.5±10.7% (mean±s.d., n=13) of the full [Ca²⁺]_er response was reached. After [Ca²⁺]_cyt reached its peak, Ca²⁺ release from the ER continued for ∼10 s (9.9±1.7 s, mean±s.d., n=13). This suggests that a significant fraction of Ca²⁺ released from the ER enters non-ER Ca²⁺ stores or exits the cell without further increasing [Ca²⁺]_cyt. The apparent delay of [Ca²⁺]_er signal amounting to ∼10 s cannot be due to the response time of the indicator, because the off rate constant of cameleon is ∼13 s⁻¹ (Miyawaki et al, 1997); indeed, there was no noticeable lag time at the onset of the first Ca²⁺ oscillation (Fig 2E,F).

The second and subsequent Ca²⁺ oscillations showed a different time course. The rate of increase in [Ca²⁺]_cyt was much lower than that of the initial spike (Fig 2D versus Fig 2E). We discerned three phases in Ca²⁺ dynamics, as indicated by the shaded boxes in Fig 2D. In phase 1 (from the bottom of [Ca²⁺]_cyt to the initiation of Ca²⁺ release from the ER), [Ca²⁺]_cyt increased, generating the foot of Ca²⁺ oscillation. Unexpectedly, [Ca²⁺]_er rather increased (that is, the ER was taking up Ca²⁺) during phase 1. As there was no entry of Ca²⁺ from the extracellular space, Ca²⁺ must be supplied from non-ER Ca²⁺ stores to generate the foot of Ca²⁺ oscillation. In phase 2 (the falling phase of [Ca²⁺]_er), ER Ca²⁺ was released, increasing [Ca²⁺]_cyt. During late phase 2, Ca²⁺ release from the ER continued, but [Ca²⁺]_cyt started to decrease after reaching a peak. This indicates that Ca²⁺ released from the ER is entering non-ER Ca²⁺ stores or leaving the cell. In phase 3 (from the bottom of [Ca²⁺]_er to the bottom of [Ca²⁺]_cyt), [Ca²⁺]_cyt decreased, whereas [Ca²⁺]_er increased, returning to the pre-oscillation level. Essentially the same patterns were detected in the presence of extracellular Ca²⁺ (Fig 2B).

The time courses of [Ca²⁺]_er and [Ca²⁺]_cyt during the second and third oscillations in different experiments were averaged after normalization. The main features of the aforementioned typical data are discernible in the averaged data, and Ca²⁺ release from the ER lags significantly after the increase in [Ca²⁺]_cyt at the onset of the third Ca²⁺ oscillation (Fig 2G).

[Ca²⁺]_cyt versus [Ca²⁺]_mt

The above results indicate that non-ER Ca²⁺ stores have an important role in Ca²⁺ dynamics during Ca²⁺ oscillations: releasing Ca²⁺ in phase 1 and absorbing Ca²⁺ in phase 2. Mitochondria function as a Ca²⁺ buffer, taking up Ca²⁺ released from the ER (Jouaville et al, 1995; Babcock et al, 1997). Thus, mitochondria may be the non-ER Ca²⁺ store involved in Ca²⁺ oscillations. We therefore examined intramitochondrial Ca²⁺ concentration ([Ca²⁺]_mt), using one of the circular permutation-type GFP-based Ca²⁺ indicators (inverse-pericam; Nagai et al, 2001) after modification. The indicator inverse-pericam2-mt carries a mitochondrial-targeting signal sequence at the amino-terminus and Citrine mutation. Calibrations using the recombinant protein at pH 8.0 (mimicking the mitochondrial environment; see Abad et al, 2004) yielded a K′_d of 80 nM (supplementary Fig 3 online). When expressed in HeLa cells, inverse-pericam2-mt was localized within tubular structures in the cytoplasm and colocalized with a mitochondrial marker dye (supplementary Fig 4 online).

Fig 3A,C shows the simultaneous measurements of [Ca²⁺]_cyt and [Ca²⁺]_mt using fura-2 and inverse-pericam2-mt, respectively, during histamine-induced Ca²⁺ oscillations in the presence and absence of extracellular Ca²⁺. There was a rapid increase in [Ca²⁺]_mt at the onset of the first [Ca²⁺]_cyt spike, and a sinusoidal change in [Ca²⁺]_mt followed in phase with the subsequent Ca²⁺ oscillations on top of the gradual decay, as reported previously (Filippin et al, 2003). With a closer look at the time course, one notices a phase difference between the two measurements. At the onset of the initial [Ca²⁺]_cyt spike, [Ca²⁺]_mt increased with [Ca²⁺]_cyt, but it continued to increase even after [Ca²⁺]_cyt reached its peak (Fig 3E). Fig 3F shows the average time courses of [Ca²⁺]_cyt and [Ca²⁺]_mt during the first Ca²⁺ oscillation after a normalization procedure similar to that in Fig 2F. Thus, at the peak [Ca²⁺]_cyt, only ∼56% (55.5±11.9%, mean±s.d., n=11) of the full [Ca²⁺]_mt response was reached. After [Ca²⁺]_cyt reached its peak, Ca²⁺ influx into mitochondria continued for 9.2±2.4 s (mean±s.d., n=11). This indicates that there is a continuous supply of Ca²⁺ into mitochondria with decreasing [Ca²⁺]_cyt. As the ER continues to release Ca²⁺ even after [Ca²⁺]_cyt reaches its peak (Fig 2E,F), the results indicate that Ca²⁺ is transferred from the ER to mitochondria during the initial Ca²⁺ spike. A similar observation has been reported by previous authors (Szabadkai et al, 2003). The in vitro calibration of inverse-pericam2-mt suggests that the peak [Ca²⁺]_mt response to histamine is of the order of several hundred nanomolar. This is lower than the previous estimations (∼3–80 μM) derived from measurements in HeLa cells using aequorin, ratiometric pericam and Rhod-2 (Collins et al, 2001; Filippin et al, 2003; Szabadkai et al, 2003), although the present estimation is similar to the value obtained in adrenal chromaffin cells (Babcock et al, 1997). There are difficulties in estimating absolute values of [Ca²⁺]_mt because of the possible uncertainties in applying in vitro calibration to in situ [Ca²⁺]_mt. Indeed, it was reported that in vitro K_d of ratiometric pericam was about ninefold lower than estimated in vivo K_d (Filippin et al, 2003).

Figure 3.

Phase difference between [Ca²⁺]_mt and [Ca²⁺]_cyt. (A) Simultaneous measurement of [Ca²⁺]_mt and [Ca²⁺]_cyt using inverse-pericam2-mt and fura-2, respectively, during histamine application in the presence of extracellular Ca²⁺ (2 mM). Data are representative of eight experiments. (B) Expanded time courses of [Ca²⁺]_mt and [Ca²⁺]_cyt of the fifth Ca²⁺ oscillation in (A). (C) [Ca²⁺]_mt and [Ca²⁺]_cyt in the absence of extracellular Ca²⁺ (2 mM EGTA). Data are representative of 11 experiments. (D) Second Ca²⁺ oscillation in (C) shown on expanded time scale. (E) Initial Ca²⁺ oscillation in (C) shown on expanded time scale. (F,G) Averaged and normalized time courses of the first Ca²⁺ oscillations (F), and the second and third Ca²⁺ oscillations (G) in the absence of extracellular Ca²⁺. Data are mean±s.e.m. (n=11).

During the second and subsequent Ca²⁺ oscillations, we noted three phases (Fig 3B,D). In phase 1′ (from the bottom [Ca²⁺]_cyt to the initiation of Ca²⁺ uptake by mitochondria), [Ca²⁺]_cyt increased gradually to form the foot of Ca²⁺ oscillation. [Ca²⁺]_mt decreased (that is, mitochondria were releasing Ca²⁺) during phase 1′. In phase 2′ (the rise phase of [Ca²⁺]_mt), [Ca²⁺]_cyt increased but reached a peak before the peak of [Ca²⁺]_mt. In late phase 2′, [Ca²⁺]_mt increased even though [Ca²⁺]_cyt decreased. In phase 3′ (from the peak [Ca²⁺]_mt to the bottom [Ca²⁺]_cyt), both [Ca²⁺]_cyt and [Ca²⁺]_mt decreased. The timing of phases 1′–3′ in mitochondrial Ca²⁺ transients well corresponded to that of phases 1–3 in ER Ca²⁺ transients. We also obtained the average time courses of [Ca²⁺]_cyt and [Ca²⁺]_mt after a normalization procedure similar to that in Fig 2G. Again, the features of the typical data are retained in the compiled data, and [Ca²⁺]_mt decreased when [Ca²⁺]_cyt began to increase in phase 1′ (Fig 3G).

Effects of interference with mitochondrial functions

The comparison of the results shown in Figs 2,3 suggests that Ca²⁺ shuttles back and forth between the ER and mitochondria, and that Ca²⁺ regulation by mitochondria is critically involved in the generation of Ca²⁺ oscillations. We further tested this possibility by disrupting the mitochondrial functions in the following experiments. We first reduced mitochondrial membrane potential using oligomycin and FCCP to inhibit Ca²⁺ uptake by the organelle. On application of histamine, [Ca²⁺]_cyt increased without oscillations (Fig 4A). [Ca²⁺]_er decreased and remained at a decreased level. [Ca²⁺]_mt hardly showed an increase. Therefore, it seems that the recycling of Ca²⁺ released from the ER is hampered in the absence of mitochondrial Ca²⁺ uptake.

Figure 4.

Importance of mitochondrial function in Ca²⁺ oscillation. (A) Effect of mitochondrial depolarizing inhibitors (oligomycin (10 μg/ml) plus FCCP (5 μM)) on histamine-induced changes in [Ca²⁺]_cyt and [Ca²⁺]_mt (upper panel), and [Ca²⁺]_cyt and [Ca²⁺]_er (lower panel). (B) Effect of mitochondrial Na⁺/Ca²⁺ exchanger inhibitor (CGP-37157, 10 μM) on histamine-induced changes in [Ca²⁺]_cyt and [Ca²⁺]_mt (upper panel), and [Ca²⁺]_cyt and [Ca²⁺]_er (lower panel). The application of mitochondrial inhibitors was initiated 10 min before the measurements. (C) During prolonged histamine application in the absence of extracellular Ca²⁺, [Ca²⁺]_mt gradually decreased and at the same time [Ca²⁺]_cyt oscillation stopped. All data were obtained in the absence of extracellular Ca²⁺, and are representative of five experiments.

We then inhibited the release of Ca²⁺ from mitochondria by inhibiting the mitochondrial Na⁺/Ca²⁺ exchanger. Following activation with histamine, Ca²⁺ is released from the ER, and both [Ca²⁺]_cyt and [Ca²⁺]_mt increased in the presence of an inhibitor of the Na⁺/Ca²⁺ exchanger, CGP-37157, as was the case under the control conditions (Fig 4B). However, the release of Ca²⁺ from mitochondria was greatly inhibited. Again, there were no Ca²⁺ oscillations and [Ca²⁺]_er remained at a decreased level. The application of CGP-37157 was initiated 10 min before the measurements, but there was no sign of significant reduction in the ER Ca²⁺ content or in the release of ER Ca²⁺ following application of histamine. This suggests that CGP-37157 had no appreciable effect on SERCA activity. We also examined the possible effect of CG-37157 on histamine-induced IP₃ production using an IP₃ indicator GFP-PHD (Hirose et al, 1999). We found no significant effect of the drug on IP₃ production (supplementary Fig 5 online).

[Ca²⁺]_mt showed sinusoidal changes, but it gradually decreased with time, probably owing to a gradual transfer of Ca²⁺ to the ER or extracellular space (Fig 3A,C). If Ca²⁺ release from mitochondria was necessary for priming each Ca²⁺ oscillation (except for the initial Ca²⁺ spike), the Ca²⁺ oscillation should have stopped after [Ca²⁺]_mt was depleted. We therefore observed Ca²⁺ oscillations in the absence of extracellular Ca²⁺ until [Ca²⁺]_mt approached the pre-stimulation level. Indeed, the Ca²⁺ oscillation pattern became irregular with the decrease in [Ca²⁺]_mt, and finally Ca²⁺ oscillation stopped (Fig 4C). Conversely, [Ca²⁺]_er between the Ca²⁺ oscillations gradually increased with time (n=13; Fig 2C). These results are consistent with the notion that mitochondrial Ca²⁺ regulation has a pivotal role in the generation of Ca²⁺ oscillations.

The model

The mechanism of the initiation of agonist-induced Ca²⁺ spikes has been studied extensively by several authors and found to show the importance of the regenerative release of Ca²⁺ from the ER (Iino et al, 1993; Bootman et al, 1997; Marchant & Parker, 2001; Miyakawa et al, 2001). The present measurements are consistent with such notion with regard to the very first Ca²⁺ oscillation, but show more features of subsequent oscillations. In the second and subsequent Ca²⁺ oscillations, [Ca²⁺]_cyt increased before the initiation of Ca²⁺ release from the ER even in the absence of extracellular Ca²⁺. Thus, we have to consider the involvement of another Ca²⁺ store. Indeed, several previous reports have indicated the role of mitochondrial in the cytoplasmic Ca²⁺ buffering and in the generation of Ca²⁺ waves and oscillations (Jouaville et al, 1995; Babcock et al, 1997; Falcke et al, 1999; also see Wagner et al, 1998). We have now directly visualized Ca²⁺ concentrations in mitochondria as well as in the ER. Our results indicate that Ca²⁺ shuttles between the ER and mitochondria along with the Ca²⁺ oscillations, and that the following sequence of events drives Ca²⁺ oscillations. The IP₃-induced Ca²⁺ release from the ER is essential for the initiation of the first Ca²⁺ spike, during which mitochondria are loaded with Ca²⁺ released from the ER (Fig 5A). During the second and subsequent Ca²⁺ oscillations, the Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ release from mitochondria supplies the primer Ca²⁺ to generate the foot of each Ca²⁺ oscillation. When the foot reaches a threshold, it then activates IP₃R, causing the regenerative release of Ca²⁺ from the ER (Fig 5B).

Figure 5.

Schematic drawings of Ca²⁺ regulation by endoplasmic reticulum and mitochondria during Ca²⁺ oscillations. (A) The first Ca²⁺ oscillation is generated by Ca²⁺ release from the endoplasmic reticulum (ER) by means of inositol 1,4,5-trisphosphate receptor (IP₃R). A considerable fraction of Ca²⁺ released from the ER is taken up by mitochondria. (B) The second and subsequent Ca²⁺ oscillations are initiated by the Ca²⁺ release from mitochondria, which then triggers regenerative Ca²⁺ release from the ER. Mitochondrial Ca²⁺ is partially reloaded. The sequence is repeated until mitochondrial Ca²⁺ is depleted. In both (A,B), the regenerative Ca²⁺-induced Ca²⁺ release mechanism underlies the upstroke of each Ca²⁺ oscillation (not shown).

It has been shown that the mitochondrial matrix buffers Ca²⁺ reversibly with an effective Ca²⁺-binding ratio of ∼4,000, which is 40 times the Ca²⁺-binding ratio of the cytoplasm (Babcock et al, 1997). Mitochondria occupy ∼12% of the volume of HeLa cells (Sesso et al, 2004). Therefore, mitochondrial Ca²⁺ is highly buffered and its release should be able to increase [Ca²⁺]_cyt. Furthermore, the inhibition of mitochondrial Ca²⁺ release either by inhibitors or by Ca²⁺ depletion interfered with Ca²⁺ oscillations (Fig 4). Taken together, both the ER and mitochondria seem to have important roles in Ca²⁺ regulation during Ca²⁺ oscillations, although we do not exclude the possibility that non-ER Ca²⁺ stores, such as the Golgi apparatus (Missiaen et al, 2004), may also make further contributions. Thus, a mitochondrial dysfunction is expected to have a significant impact on Ca²⁺ oscillation dynamics.

Methods

Indicators. The complementary DNA fragments encoding CFP-CaM7-er, M13-Citrine-er and inverse-pericam2 were constructed on the basis of pcDNA3-YC4.1er and pcDNA3-inverse-pericam (gift from Dr A. Miyawaki, RIKEN, Wako, Japan). Details of mutagenesis, generation of recombinant proteins and retroviruses are available in the supplementary information online.

Fluorescence imaging. HeLa cells transduced with retroviruses encoding the indicators and cultured in glass-bottomed dish (MatTek) were loaded at room temperature (23°C–25°C) with 5 μM indo-5F AM or fura-2 AM in physiological salt solution containing 150 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose and 25 mM Hepes (pH 7.4). Pairs of fluorescence images at 480 and 535 nm were simultaneously captured using an inverted microscope (IX70; Olympus, Japan) equipped with a × 60 objective (NA 1.2) and an image acquisition system (W-View; Hamamatsu Photonics, Japan) at a rate of one frame per 2 s. The excitation wavelengths were 346 nm for indo-5F, 434 nm for split-YC7.3er cameleon, 380 nm for fura-2 and 490 nm for inverse-pericam2-mt.

For the simultaneous imaging of [Ca²⁺]_cyt (indo-5F) and [Ca²⁺]_er (split-YC7.3er cameleon), fluorescence images at 480 nm from indo-5F with 346 nm excitation represent [Ca²⁺]_cyt, and those at 535 nm fluorescence were divided by the corresponding images at 480 nm on a pixel-to-pixel basis to obtain the fluorescent resonance energy transfer efficiency of split-YC7.3er cameleon at 434 nm excitation. For the simultaneous imaging of [Ca²⁺]_cyt (fura-2) and [Ca²⁺]_mt (inverse-pericam2-mt), fluorescence images at 535 nm from either inverse-pericam2-mt (490 nm excitation) or fura-2 (380 nm excitation) were alternately obtained.

Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400620-s1.pdf).

Supplementary Material

Supplementary Information