The endoplasmic reticulum (ER) and mitochondria are membrane-bound intracellular organelles in eukaryotic cells that carry out vital functions. Both ER and mitochondrial membranes display Ca2+-transporting molecules whose function is to import Ca2+ into the lumen against the concentration gradient. This uphill Ca2+ transport is mediated in the ER membrane by sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)1 and in the inner mitochondrial membrane by the mitochondrial Ca2+ uniporter (MCU)2. The organellar membranes also feature molecules that allow Ca2+ to exit from the organelles to the cytosol: inositol 1,4,5-trisphosphate receptors and ryanodine receptors in the ER, and the Na+/Ca2+ and H+/Ca2+ exchangers in mitochondria2 3. Thus, the intraluminal Ca2+ concentration in the organelles is tightly regulated, and may exceed that of cytosol by a large factor. Ca2+ levels within the ER profoundly affect organelle function, and overload or depletion causes ER stress4. Within the mitochondrial matrix, Ca2+ concentration regulates the rate of ATP production, and abnormal concentrations can lead to cell death or autophagic degradation of the mitochondria2. ER and mitochondrial structures are constantly being reorganized and close contacts form between the two types of organelles. These contact sites have recently been shown to be involved in diverse functions, including lipid biosynthesis, mitochondrial biogenesis and the transfer of Ca2+ (refs 5, 6, 7, 8). Both types of organelle are also involved in the regulation of cytosolic Ca2+ concentrations. Release of Ca2+ from the ER regulates contraction, fertilization, development, secretion and synaptic plasticity3. In addition, the ER luminal Ca2+ concentration regulates Ca2+ influx across the plasma membrane. Following a release of Ca2+ from the ER, STIM1, which is present in the ER membrane, functions as a Ca2+ transducer; it signals to the plasma membrane to activate the store-operated Ca2+ entry mechanism (SOCE)9 10. SOCE is mediated by the molecular complex that includes Orai1, and is important for the activation of various cell functions, the best-studied example of which is the immune responses9. In contrast to the active role of the ER in Ca2+ signalling, mitochondria have been considered to act as a passive Ca2+ buffer2. However, recent results suggest that they may also have an active role as a source of Ca2+ in the regulation of cytosolic Ca2+ concentrations11. Although the importance of the ER and mitochondria as Ca2+-handling organelles is unequivocal, the mechanism by which organellar Ca2+ concentrations regulate cellular processes remains elusive. New methods to dissect organellar Ca2+ dynamics are expected to facilitate such studies. While small molecular Ca2+ indicators cannot be precisely targeted to the organelles, limiting their use in living cells, genetically encoded Ca2+ indicators (GECIs) can be targeted to organelles with the addition of appropriate tags12. Making use of this capability, GFP-based GECIs13 14 15 16 17 18 19 20 21 22 23 and aequorin (a Ca2+-sensitive photoprotein)24 have been used to measure intraorganellar Ca2+ concentrations. FRET-type GFP-based GECIs were first used to measure intraluminal Ca2+ concentration in the ER and were applied to different cell types13 18 19 21 22 23. This type of indicators uses wide visible wavelength bands for excitation and emission, often limiting the simultaneous use of other fluorescent molecules25 26. Subsequently, innovative modifications of the GFP molecule have yielded single-wavelength-excitation GECIs with various affinities to Ca2+ for ER and mitochondrial Ca2+ imaging14 15 16 17 20. Aequorin emits dim light, and simultaneous measurement with brighter fluorescence signals is not possible with most fluorescence microscopes. Although we have a wide variety of indicators, simultaneous Ca2+ imaging of the ER and mitochondria has not been carried out, and improvement in the spatiotemporal resolution of organellar Ca2+ is expected to enhance our understanding of intraorganellar Ca2+ dynamics. For these reasons, and to study the functional interaction between the ER and mitochondria, a new type of GECIs with higher spatiotemporal resolution was required. This study reports on the generation of new organellar Ca2+ indicators that allow simultaneous imaging of two subcellular compartments with high spatiotemporal resolution. They are optimized in terms of Ca2+ affinity and dynamic range for organellar Ca2+ imaging and come in colour variants for simultaneous measurement of multiple signals when they are used in appropriate combinations. Using them, intraorganellar Ca2+ concentrations can be imaged at unprecedented spatiotemporal resolution. To illustrate the utility of the approach, we demonstrate high spatiotemporal resolution imaging of ER and mitochondrial Ca2+ dynamics in living cells; we show the quantitative relationship between the ER Ca2+ concentration and the extent of STIM1 puncta formation in the regulation of SOCE; and we show that inhomogeneity in mitochondrial Ca2+ responses can be observed during apparently homogenous ER and cytosolic Ca2+ changes, which suggest that there is a mechanism to regulate the influx of Ca2+ into mitochondria. The new indicators described in this work will be valuable for further study of the roles of the ER and mitochondria, and of their functional interactions. Results Development of ER Ca2+ indicator We began measuring ER Ca2+ dynamics based on a lead variant of GCaMP2 (cfGCaMP2, see Methods), whereby fluorescence intensity increased 5.1-fold upon binding of Ca2+ with a K d of 0.67 μM (Supplementary Table 1). Since ER Ca2+ concentration ([Ca2+]ER) is assumed to reach the sub-millimolar range, cfGCaMP2 was engineered to reduce its Ca2+ binding affinity by a factor of ~1,000. We searched for low Ca2+ affinity variants guided by extensive structure–function analyses based on site-directed mutagenesis in the calmodulin domain (See Methods). From among the 58 variants that were generated in our search we selected one with E31D/F92W/E104D/D133E substitutions that had a low Ca2+ affinity (K d=368 μM) and a large dynamic range (F max/F min=4.2) (Fig. 1a and Supplementary Fig. 1a,b). After attaching ER localization and retention signal sequences, the low Ca2+ affinity variant was expressed in HeLa cells. The engineered indicator protein colocalized with an ER marker27 (Supplementary Fig. 1c). Upon addition of thapsigargin, an inhibitor of SERCA, a large reduction in fluorescence intensity was noted (Fig. 1c). Also, oscillatory fluorescence intensity decreased in response to histamine, which generates cytosolic Ca2+ oscillations due to release of Ca2+ from the ER via IP3Rs (Fig. 1d). Thus, the indicator successfully reports [Ca2+]ER dynamics. We designated it Calcium-measuring organelle-Entrapped Protein IndicAtor 1 in the ER (CEPIA1er). Multi-coloring of CEPIA We generated colour variants of CEPIA, based on recently developed cytosolic Ca2+ indicators: R-GECO1 (red fluorescence), G-GECO1.1 (green fluorescence), and GEM-GECO1 (ratiometric blue/green fluorescence)20. After rigorous structure–function assessment (see Methods), we obtained R-CEPIA1er (K d=565 μM, F max/F min=8.8), G-CEPIA1er (K d=672 μM, F max/F min=4.7), and GEM-CEPIA1er (K d=558 μM, R max/R min=21.7; Fig. 1a and Supplementary Table 1). In vitro characteristics of these CEPIA variants, compared with those of original GECO, are summarized in Table 1 and Supplementary Fig. 2a–d. We expressed the colour variants of CEPIA with ER-targeting signal sequences in HeLa cells. They localized to the ER27 28, and detected ER Ca2+ oscillations with a high signal-to-noise ratio (Fig. 1b–e and Supplementary Fig. 3a,b). In HeLa cells, G-CEPIA1er showed superior performance in signal amplitude over CEPIA1er (Fig. 1d). To examine whether pH changes had any effect on the signal of CEPIA indicators (Supplementary Fig. 2c), we monitored pH dynamics in the ER using a pH sensor, enhanced yellow fluorescent protein (EYFP)29 30. There was no significant change in the fluorescence intensity of ER-localized EYFP (Supplementary Fig. 4a–d), verifying that the CEPIA responses are not due to pH changes in the ER. To measure the Ca2+ affinity of CEPIA variants within the ER, we carried out Ca2+ titration experiments in permeabilized HeLa cells. Stepwise changes in Ca2+ concentration elicited dose-dependent fluorescence intensity changes in the presence of ionomycin to make the ER membrane permeable to Ca2+ (Supplementary Fig. 3c). The K d values determined within the ER were almost equivalent to those measured in vitro (Supplementary Fig. 3d). Thus, we succeeded in expanding the hues of CEPIA variants. Estimation of [Ca2+]ER Using ratiometric measurement of GEM-CEPIA1er, we estimated [Ca2+]ER in intact resting cells as varying between 620 and 860 μM in HeLa cells, HEK293A cells, BHK cells and cultured astrocytes; this decreased to 310–570 μM upon stimulation with agonists (Fig. 1f). The range of [Ca2+]ER underlines the need to increase the indicator’s K d to >100 μM to provide faithful measurements of ER Ca2+ dynamics, and explains the difficulty in imaging ER Ca2+ dynamics using D1ER, which has a K d of ~60 μM (ref. 19; Fig. 1a,c,d). Although CEPIA indicators had relatively high Hill coefficients (n=1.4–2.0, Table 1), the relationship between ΔF/F max (or ΔR/R max) and changes in [Ca2+] is not highly distorted within the physiological [Ca2+]ER range (Supplementary Fig. 2e). Subcellular ER Ca2+ dynamics visualized with CEPIA We next examined whether CEPIA indicators are capable of detecting ER Ca2+ dynamics at subcellular resolution. Agonists often induce Ca2+ waves, which propagate throughout the cell after initiation in focal regions31 32. The wave is generated by the regenerative release of Ca2+ from the ER, and it has been predicted that this mechanism creates an ‘inverse Ca2+ wave’ within the ER. However, this prediction has not been tested using GECIs in live cells. We used G-CEPIA1er imaging at a high frame rate (10–30 frames s–1) to visualize inverse Ca2+ waves in the ER. Local decreases in [Ca2+]ER were observed, initiating at the tips and propagating to the perinuclear region in HeLa cells (Fig. 2a and Supplementary Movie 1). The time courses measured at two subcellular locations indicated a wave-like propagation of decreasing [Ca2+]ER (Fig. 2b,c). The speed of these waves was 60.8±3.2 μm s–1 (mean±s.e.m.), which matches cytosolic Ca2+ waves with or without CEPIA expression (Fig. 2d). Similar observations were made with R-CEPIA1er. Thus, CEPIA indicators have high spatiotemporal resolution. We next examined whether CEPIA can be applied to intact tissue preparations. To do so, Ca2+ dynamics were elucidated in the neuronal ER in response to synaptic inputs to Purkinje cell dendrites in cerebellar slice preparations. G-CEPIA1er was expressed in Purkinje cells by Sindbis virus and was imaged with a two-photon microscope. G-CEPIA1er expression was observed throughout the dendrites and into spines, matching the distribution of the ER in Purkinje cells (Fig. 3a). In response to parallel fibre stimulation, which induces Ca2+ release from the ER by activating the metabotropic glutamate receptor33, we observed a long-lasting decrease in G-CEPIA1er fluorescence intensity (Fig. 3b and Supplementary Movie 2). The ER Ca2+ dynamics could be visualized at the level of single spines (Fig. 3c). This response was not due to any pH change in the ER, because parallel fibre stimulation had no effect on pH within the ER measured by ER-targeted EYFP (Supplementary Fig. 4e–g)29 30. Thus, subcellular ER Ca2+ imaging using CEPIA is applicable to tissue preparations that have retained three-dimensional structure, which often requires two-photon excitation. Simultaneous imaging of Ca2+ dynamics in the ER and cytosol For simultaneous imaging of Ca2+ dynamics in the ER and cytosol, we used the ratiometric small-molecule Ca2+ indicator, fura-2 (excitation: 340–380 nm), together with G-CEPIA1er or R-CEPIA1er. ER Ca2+ signals were a mirror image of the cytosolic Ca2+ oscillations (Supplementary Fig. 5a,b). Although G-CEPIA1er and R-CEPIA1er are weakly excited at the excitation wavelengths of fura-2 (Supplementary Figs 2a and 6a), they had very little effect on the fura-2 fluorescence ratio (Supplementary Fig. 6b). Thus, it is possible to use G-CEPIA1er and R-CEPIA1er with fura-2. Either G-CEPIA1er or GEM-CEPIA1er can be co-expressed with R-GECO1 for simultaneous imaging of the ER and cytosolic Ca2+ dynamics (Supplementary Fig. 5c–f and Supplementary Movie 3). Reversal of the colours is also possible, and ER Ca2+ imaging using R-CEPIA1er can be simultaneously carried out with cytosolic Ca2+ imaging using G-GECO1.1 or GEM-GECO1 (Supplementary Fig. 5g,h). Spectral bleed-through was minimal ( 95% purity was prepared by several replatings of the brain cell culture, which was obtained by 2.5% trypsin treatment and gentle trituration of the neocortices or the hippocampi of embryonic days 18–19 or postnatal days 1–3 Sprague–Dawley rat foetuses66. For Ca2+ imaging, the cells were plated on collagen type-I-coated glass-bottom dishes (MatTek, USA) or Cell-Tak-coated dishes (Corning, USA) before imaging. Time-lapse Ca2+ imaging Cultured cells were transfected using Lipofectamine 2000 (Invitrogen) 2 or 3 days before imaging. Jurkat T cells were electroporated using a MicroPorator (MP-100, Digital Bio) 1 day before imaging. For cytosolic Ca2+ imaging using fura-2, cells were loaded with 5 μM fura-2 AM (Molecular Probes, USA) at room temperature (22–24 °C) for 40–60 min in 0.1% BSA-supplemented physiological salt solution (PSS) containing (in mM) 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5.6 glucose and 25 HEPES (pH 7.4). Before imaging, the loading solution was replaced with PSS without BSA. The images were captured using an inverted microscope (IX81, Olympus, Japan) equipped with a × 20 objective (numerical aperture (NA)=0.75, UPlanSApo, Olympus) or a × 40 objective (NA 0.90, UApo/340, Olympus), an electron-multiplying cooled-coupled device (EM-CCD) camera (ImagEM, Hamamatsu Photonics, Japan), a filter wheel (Lambda 10-3, Sutter Instrument, USA), a xenon lamp (ebx75) and a metal halide lamp (EL6000, Leica, Germany) at a rate of one frame per 2 or 3 s with the following excitation/emission filter settings: 472±15 nm/520±17.5 nm for G-GECO1.1, CEPIA1er, G-CEPIA1er, CEPIA2–4mt and EYFP-er; 562±20 nm/641±37.5 nm for R-GECO1, R-CEPIA1er and mCherry-STIM1; 377±25 nm/466±20 nm and 377±25 nm/520±17.5 nm for GEM-GECO1 and GEM-CEPIA1er; 340±13 nm/510±42 nm and 365±6 nm/510±42 nm for fura-2; 440±10.5 nm/480±15 nm and 440±10.5 nm/535±13 nm for D1ER19 20. For analysis of the ratiometric indicators, we calculated the fluorescence ratio (F 466/F 520 for GEM-GECO1 and GEM-CEPIA1er; F 340/F 365 for fura-2; F 535/F 480 for D1ER). Photobleaching was corrected for using a linear fit to the fluorescence intensity change before agonist stimulation. All images were analysed with ImageJ software. To image subcellular ER Ca2+ dynamics during agonist-induced Ca2+ wave formation, we imaged HeLa cells expressing either G-CEPIA1er or R-CEPIA1er. Images were captured at a rate of one frame per 30–100 ms using a × 60 objective (NA 1.45, PlanApo TIRF, Olympus) and the metal halide lamp or an LED lamp (pE-100, CoolLED, UK). To evaluate Ca2+ wave velocity in the ER and cytosol, images were normalized by the resting intensity, and a linear region of interest (ROI) was defined along the direction of wave propagation. A line-scan image was created by averaging 30 adjacent linear ROIs parallel to the original ROI, and time derivative was obtained to detect the time point that showed maximal change during the scan duration. Then, the time points were plotted against the pixel, and the wave velocity was estimated by the slope of the least-squares regression line. For mitochondrial Ca2+ imaging with ER and cytosolic Ca2+, mitochondrial inner membrane potential or mitochondrial pH at subcellular resolution, we imaged HeLa cells with a confocal microscope (TCS SP8, Leica) equipped with a × 63 objective (NA 1.40, HC PL APO, Leica) at a rate of one frame per 2 or 3 s with the following excitation/emission spectra: R-GECO1mt (552 nm/560–800nm), G-CEPIA1er (488 nm/500–550 nm) and GEM-GECO1 (405 nm/500–550 nm); GEM-GECO1mt (405 nm/500–550 nm), JC-1 (488 nm/500–550 nm and 488 nm/560–800nm); R-GECO1mt (552 nm/560–800nm), SypHer-dmito (405 nm/500–550 nm and 488 nm/500–550 nm). For analysis of JC-1 and SypHer-dmito, we calculated the fluorescence ratio (488 nm/560–800 nm over 488 nm/500–550 nm for JC-1 (ref. 55); 488 nm/500–550 nm over 405 nm/500–550 nm for SypHer-dmito62). To perform in situ Ca2+ titration of CEPIA, we permeabilized the plasma membrane of HeLa cells with 150 μM β-escin (Nacalai Tesque, Japan) in a solution containing (in mM) 140 KCl, 10 NaCl, 1 MgCl2 and 20 HEPES (pH 7.2). After 4 min treatment with β-escin, we applied various Ca2+ concentrations in the presence of 3 μM ionomycin and 3 μM thapsigargin, and estimated the maximum and minimum fluorescent intensity (R max and R min), dynamic range (R max/R min), K d and n. For the estimation of [Ca2+]ER based on the ratiometric measurement using GEM-CEPIA1er (Figs 1e,f and 5b and Supplementary Fig. 5f), [Ca2+]ER was obtained by the following equation: where R=(F at 466 nm)/(F at 510 nm), n=1.37 and K d=558 μM. To evaluate pH-dependent change of EYFP-er fluorescence (Supplementary Fig. 4a–d), we stimulated HeLa cells expressing EYFP-er in a PSS (adjusted to pH 6.8) containing monensin (10 μM, Wako) and nigericin (10 μM, Wako). Subsequently, the cells were alkalinized with a solution containing (in mM) 120 NaCl, 30 NH4Cl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES and 5.6 Glucose (pH 7.4)67. Analysis of STIM1 dynamics STIM1 dynamics were analysed using the ImageJ software. To extract STIM1 puncta, the captured images were binarized after filtering (Gaussian Blur or Unsharp Mask). Then, particles greater than 2 pixels (corresponding to ~1.2 μm2) were counted using the ‘Analyse Particles’ tool in ImageJ. The counts were normalized by the maximum values (N puncta) and plotted against the ER Ca2+ levels (ER Ca2+) ([Ca2+]ER for GEM-CEPIA1er and F/F 0 for G-CEPIA1er). Curve fitting was performed using the following Hill equation: where K 1/2 indicates the ER Ca2+ level at half-maximal puncta formation and n represents Hill coefficient68. Imaging of subcellular localization of CEPIA All images were captured with a confocal microscope (TCS SP8) equipped with a × 63 objective at the following excitation/emission wavelengths: CEPIA1er, G-CEPIA1er, CEPIA2mt, EGFP-er and EYFP-er (488 nm/500–550 nm), R-CEPIA1er, mCherry-er and MitoTracker Red (Invitrogen; 552 nm/560–800nm) and GEM-CEPIA1er (405 nm/420–550 nm). The obtained images were merged with ImageJ software. ER Ca2+ imaging in cerebellar slices For Purkinje cell-specific expression of G-CEPIA1er, we produced a Sindbis virus vector. The pSinRep5-G-CEPIA1er vector was then used as the template for in vitro transcription using SP6 RNA polymerase (Ambion, USA). The RNA transcript and the helper RNA from DH(26S) cDNA template (Invitrogen) were cotransfected into BHK cells by electroporation. Twenty-four hours after transfection, the culture medium containing the infectious particles was harvested. Sindbis virus vector encoding EYFP-er was also produced with the same procedure. C57BL/6 mice (postnatal days 21–27) were deeply anaesthetized with pentobarbital, and the surface of cerebellar lobule 6 beside the midline was exposed by removing the cranium and dura. The tip of a glass pipette was backfilled with the viral solution. The glass pipette was then inserted into the cerebellum and 1 μl of viral solution was delivered at a rate of 200 nl min–1 using a micropump (Legato 130, KD Scientific, USA). Twenty-four hours after virus injection, parasagittal cerebellar slices (250 μm thickness) were prepared69. Mice were anaesthetized with diethyl ether and decapitated. The brain was removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) bubbled with 95% O2/5% CO2. Slices were cut using a microslicer (PRO 7, Dosaka EM, Japan). The slices were incubated in a holding chamber containing ACSF bubbled with 95% O2 and 5% CO2 at 35 °C for 1 h and then returned to 23 °C. ACSF for slicing and incubation contained (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3 and 20 glucose. Slices were transferred to a recording chamber under a microscope, and continuously perfused with ACSF supplemented with 10 μM bicuculline (Tocris Bioscience, UK) and 10 μM NBQX (Tocris Bioscience) to block inhibitory postsynaptic potentials and accompanying Ca2+ influxes. ACSF was bubbled with 95% O2 and 5% CO2. Imaging was carried out with a two-photon microscope (TSC MP5, Leica) equipped with a water-immersion objective ( × 25, NA 0.95, HCS IR APO, Leica) and a Ti:sapphire laser (MaiTai DeepSee; Spectra Physics, USA). Excitation wavelength was 900–920 nm for both G-CEPIA1er and EYFP-er. Data were acquired with time-lapse XY-scan mode (8 Hz) and analysed using ImageJ software. Fluorescence intensities were corrected for background fluorescence by measuring a non-fluorescent area. When necessary, photobleaching was corrected for using a linear fit to the fluorescence intensity change. For the focal stimulation of parallel fibres, square pulses (0.1 ms) were applied through stimulation pipettes (3–6 μm tip diameter) filled with ACSF. The stimulation intensity was adjusted within 4–5 V to induce G-CEPIA1er signals with a range of ~20 μm in diameter. For EYFP-er, the stimulation intensity was fixed at 5 V. Experiments were carried out at room temperature. Statistics Two-tailed Student’s t-tests were performed to determine the significance if not stated otherwise. Author contributions J.S. and Y.O. performed experiments and analysed the data; J.S., K.K., Y.O. and M.I. designed the experiment. J.S., K.K. and M.I. wrote the paper; K.I. and M.O. generated the cfGCaMP2. Additional information How to cite this article: Suzuki, J. et al. Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat. Commun. 5:4153 doi: 10.1038/ncomms5153 (2014). Supplementary Material Supplementary Information Supplementary Figures 1-8, Supplementary Tables 1-2. Supplementary Movie 1 Wave-like propagation of Ca2+ release from the ER visualized with G-CEPIA1er. Duration is 9-s with frames captured at 35-ms intervals. The time courses of this movie are shown in Fig. 2c. Supplementary Movie 2 Activity-dependent ER Ca2+ dynamics in cerebellar Purkinje cells visualized with G-CEPIA1er. Duration is 10 sec with frames captured at 125-ms intervals. The time courses of this movie are shown in Fig. 3b. Supplementary Movie 3 Simultaneous Ca2+ imaging in the ER and cytosol visualized with G-CEPIA1er and R-GECO1. Duration is 5 min with frames captured at 2-s intervals. The time courses of this movie are shown in Supplementary Fig. 5c. Supplementary Movie 4 Simultaneous imaging of STIM1 localization and ER Ca2+ dynamic visualized with mCherry-STIM1 and GEM-CEPIA1er. Duration is 14 min with frames captured at 20-s intervals. The time courses of this movie are shown in Fig. 5b. Supplementary Movie 5 Localized Ca2+ signal in mitochondria visualized with CEPIA3mt. Duration is 1.5 min with frames captured at 1-s intervals. The time courses of this movie are shown in Fig. 7b. Supplementary Movie 6 Simultaneous Ca2+ imaging in the mitochondria, ER and cytosol during localized Ca2+ signal in mitochondria visualized with R-GECO1mt, G-CEPIA1er and GEM-GECO1. Duration is 1.2 min with frames captured at 3-s intervals. The time courses of this movie are shown in Fig. 7e.
