Mitochondria Recycle Ca2 to the Endoplasmic Reticulum and Prevent

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 31, Issue of August 3, pp. 29430 –29439, 2001 Printed in U.S.A.

Mitochondria Recycle Ca2ⴙ to the Endoplasmic Reticulum and Prevent the Depletion of Neighboring Endoplasmic Reticulum S Regions*□ Received for publication, April 12, 2001, and in revised form, May 14, 2001 Published, JBC Papers in Press, May 17, 2001, DOI 10.1074/jbc.M103274200

Serge Arnaudeau‡, William L. Kelley§, John V. Walsh, Jr.¶, and Nicolas Demaurex‡储 From the ‡Department of Physiology, University of Geneva, 1211 Geneva 4, Switzerland, the §Division of Infectious Diseases, Geneva University Hospitals, 1211 Geneva 4, Switzerland, and the ¶Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

To study Ca2ⴙ fluxes between mitochondria and the endoplasmic reticulum (ER), we used “cameleon” indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in ⬃20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 ⴞ 5 ␮M, indicating that Ca2ⴙ transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca2ⴙ]ER averaged 371 ⴞ 21 ␮M at rest and decreased to 133 ⴞ 14 ␮M and 59 ⴞ 5 ␮M upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca2ⴙ uptake was prevented by oligomycin and rotenone or when Ca2ⴙ efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca2ⴙ taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca2ⴙ than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca2ⴙ uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca2ⴙ release and recycle a substantial portion of the captured Ca2ⴙ back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca2ⴙ signals and the filling state of neighboring ER regions.

The calcium ion is a ubiquitous intracellular messenger that controls processes ranging from fertilization and cellular differentiation to muscle contraction and synaptic transmission (1, 2). The finely regulated spatial and temporal encoding of the calcium signal ensures that these various, and sometimes opposite, calcium-dependent processes are activated at the appropriate time and place within cells (3–5). Whereas the influx of * This work was supported by operating Grants 31-46859.96 and 31-55344.98/1 from the Swiss National Science Foundation and in part by Grant HL61297 (to J. W.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains videos (Fig. 4). 储 Fellow from the Prof. Dr. Max Cloe¨tta Foundation. To whom correspondence should be addressed: Dept. of Physiology, University of Geneva Medical Center, 1, Michel-Servet, CH-1211 Geneva 4, Switzerland. Tel.: 4122-7025399; Fax: 4122-702-5402; E-mail, [email protected].

calcium through voltage-dependent membrane channels triggers rapid secretion at synapses (6), the release of Ca2⫹ from stores in response to inositol 1,4,5-trisphosphate (IP3)1 can generate sustained [Ca2⫹]cyt oscillations in both excitable and nonexcitable cells (7). These calcium oscillations can be decoded in the cytosol by frequency-sensitive effector proteins such as calmodulin-dependent kinase II (8), and have been shown to optimize both secretion (9) and gene expression (10, 11). Calcium oscillations can also be decoded by mitochondria (12), several dehydrogenases being activated as the free [Ca2⫹] increases within the mitochondrial matrix, thereby increasing the level of NAD(P)H and the production of ATP to meet the cell energy demand (12, 13). In addition to being able to decode Ca2⫹ oscillations, mitochondria also participate actively in calcium signaling (reviewed in Refs. 14 –16). Mitochondria take up calcium very efficiently and contribute to the local nature of the calcium signal by acting as a buffer barrier between cellular regions (17). Importantly, mitochondria are often in close contact with Ca2⫹ release sites in the endoplasmic reticulum (18, 19) or with Ca2⫹ influx channels at the plasma membrane (20). By acting as a Ca2⫹ buffering system at these strategic locations, mitochondria can modulate the rate of Ca2⫹ release by IP3 receptors (21, 22) or the rate of capacitative Ca2⫹ entry through CRAC channels (20). Through this intimate connection with the calcium sources, mitochondria strongly shape calcium signals and, depending on the cellular context, can either potentiate or inhibit Ca2⫹ oscillations (23–26). Our understanding of the calcium homeostasis of intracellular compartments is still incomplete, because the highly dynamic Ca2⫹ signals occurring within organelles are difficult to measure. Trapped fluorescent dyes such as Mag-fura and Magindo-1 have been used to measure calcium within the ER and mitochondria (27–31). However, these dyes are not specifically targeted and their selectivity for calcium over magnesium is poor. The cationic probe rhod2 has also been used to measure calcium within mitochondria (12, 20, 21, 24) but its specificity relies on the negative membrane potential of this organelle. The calcium-sensitive photoprotein aequorin, on the other hand, can be specifically targeted (32, 33) and has been used extensively to measure calcium dynamics within the mitochondria (13, 34 –36), the ER (37– 41), and the Golgi complex (42). 1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; [Ca2⫹]cyt, cytosolic free Ca2⫹ concentration; [Ca2⫹]ER, endoplasmic reticulum free Ca2⫹ concentration; [Ca2⫹]mit, mitochondrial matrix free Ca2⫹ concentration; CGP 37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; TG, thapsigargin; SERCA, sarco/endoplasmic reticulum Ca2⫹-ATPase; YC, yellow cameleon; EYFP, enhanced yellow fluorescent protein.

29430

This paper is available on line at http://www.jbc.org

Calcium Cycling between the ER and Mitochondria

29431

selective targeting sequences, allows one to visualize the calcium signals in organelles by fluorescence ratio imaging (45– 47). Furthermore, the calcium affinity of calmodulin can be adjusted by molecular engineering, enabling one to match the calcium concentration within the organelle of interest (43). Despite these advantages, the cameleons have not yet found widespread applications, probably because their limited dynamic range and pH dependence requires careful in situ calibration to achieve quantitative Ca2⫹ measurements. In this study, we used yellow cameleons Ca2⫹ indicators to measure Ca2⫹ signals in the cytosol, [Ca2⫹]cyt, the endoplasmic reticulum, [Ca2⫹]ER, and the mitochondria, [Ca2⫹]mit in HeLa cells. Probes of different Ca2⫹ affinities and pH dependence were used (YC2, YC4ER, YC2mit, YC3.1mit, and YC4.1mit) and calibrated in situ, providing accurate estimates of the free Ca2⫹ concentration within the ER lumen and the mitochondrial matrix. Using this approach, we show that [Ca2⫹]mit transients reach ⬎100 ␮M in about 25% of mitochondria, and that part of the captured Ca2⫹ is returned back to the ER. The local cycle of Ca2⫹ between these two organelles prevents the depletion of ER regions bearing mitochondria, thereby generating two functionally distinct Ca2⫹ stores within the ER network. EXPERIMENTAL PROCEDURES

FIG. 1. Staining patterns and subcellular localization of the cytosolic, ER, and mitochondrial probes. a, typical fluorescence (430 ⫾ 10 nm excitation, 535 ⫾ 12.5 nm emission) of intact HeLa cells transfected with the YC2 probe. Images are shadow projections of 21 adjacent, 250 nm wide z sections deconvoluted with the iterative constrained Tikhonov-Miller restoration algorithm. b, left: HeLa cells transiently transfected with ER-targeted YC4ER probe. Right, the cells were fixed, permeabilized, and stained with an anti-calreticulin antibody; the YC4ER fluorescence co-localized with the calreticulin immunostaining. c, left: HeLa cells expressing YC2mit. Right, cells expressing the YC3.1mit probe were co-labeled with the vital dye mitotracker red to assess the specificity of the mitochondrial probe. Size bar: 5 ␮m.

However, the weak luminescence of the photoprotein and its irreversible consumption upon Ca2⫹ binding severely limits the use of this approach for calcium imaging. The “cameleon” indicators based on green fluorescent proteins and calmodulin developed in the group of R. Y. Tsien (43, 44) appear better suited for calcium measurements in organelles. The bright fluorescence of the green fluorescent protein mutants, combined with

Materials—Dulbecco’s modified Eagle’s culture medium, fetal calf serum, penicillin, streptomycin, geneticin were obtained from Life Technologies, Inc. (Paisley, Scotland). Histamine, epinephrine, thapsigargin, nigericin, monensin, ATP, and HEPES were purchased from Sigma. Ionomycin, EGTA, and HEEDTA were obtained from Fluka (Buchs, Switzerland). CGP 37157 was from Tocris (Bristol, United Kingdom). Transfast transfection reagent was purchased from Promega (Catalys AG, Switzerland). All other chemicals were of analytic grade and were obtained from Fluka or Sigma. The “Ca2⫹ medium” contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 20 mM Hepes, pH 7.4. The “Ca2⫹-free medium” contained no CaCl2 and 0.5 mM EGTA. Drugs were dissolved in dimethyl sulfoxide or ethanol and diluted in the recording medium on the day of use, at a final solvent concentration ⬍0.1%. Constructs—Plasmids YC2, YC2.1, and YC4ER were kindly provided by Dr. R. Y. Tsien. Plasmid YC2mit was generated by ligation of the NotI YC2 insert into pCMV/myc/Mito (Invitrogen). Plasmid YC2.1mit was then generated by exchanging the EYFP fragments of YC2.1 and YC2mit using SacI and BstX1. Plasmids YC3mit, YC3.1mit, YC4mit, and YC4.1mit were prepared by the Quick Change method using Pfu polymerase (Stratagene) and YC2mit or YC2.1mit as templates. Complementary primer pairs: E31Q-A: 5⬘-gacggcaccatcaccacaaagcagctgggcaccgttatgaggtcgc-3⬘; E31Q-B: 5⬘-gcgacctcataacggtgcccagctgctttgtggtgatggtgccgtc-3⬘; E104Q-A: 5⬘-aacggctacatcagcgctgctcagctgcgtcacgtcatgacaaacc-3⬘; E104Q-B: 5⬘-ggtttgtcatgacgtgacgcagctgagcagcgctgatgtagccgtt-3⬘, were designed for each mutant E31Q or E104Q in the calmodulin module. For convenience, a PvuII restriction site was introduced (underlined) with each mutant by simultaneously changing the adjacent leucine codon L32 or L105 to an alternative codon with high relative abundance in mammalian usage tables. Following 12 PCR cycles with annealing at 60 °C and extension at 68 °C, products were digested with DpnI, then electroporated into strain JM109. Transformants were first screened by NotI and PvuII digestion. The entire cameleon coding sequence for each mutant was then verified using dideoxy dye termination sequencing and appropriate primers. Plasmid DNAs were purified using a Qiagen column by the Maxi-prep purification protocol recommended by the manufacturer. Cell Culture and Transfection—HeLa and HEK-293 cells (purchased from American type Culture Collection, Rockville, MD) were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 ␮g/ml streptomycin, and were maintained in a humidified incubator at 37 °C in the presence of 5% CO2, 95% air. Cells (⬃200,000) were plated on 25-mm glass coverslips. After they reached 60% of confluence, cells were transiently transfected with cDNAs encoding the yellow cameleons probes, using the calcium phosphate protocol for HEK-293 cells or Transfast reagent for HeLa cells. Cells were imaged 3 to 5 days after transfection. Stable HEK-293 transfectants were grown in the presence of geneticin (100 ␮g/ml) for 3 weeks and ⬃20 clones were expanded for each condition and tested for

29432

Calcium Cycling between the ER and Mitochondria Fluorescence emission from the cameleons was imaged using a cooled 12-bit CCD interlined camera (Visicam, Visitron System, Germany) at two emission wavelengths, using a filterwheel (Ludl Electronic Products, Hawthorn, NY) to alternatively change the two emission filters (475DF15 and 535DF25, Omega Optical, Brattleboro, VT). Image acquisition and analysis was performed with the Metamorph/ Metafluor 3.5 software (Universal Imaging, West Chester, PA). Images were stored on optical discs for later analysis and archiving. Changes in fluorescence ratio R ⫽ (fluorescence intensity at 535 nm ⫺ background intensity at 535 nm)/(fluorescence intensity at 475 nm ⫺ background intensity at 475 nm) were calibrated in [Ca2⫹] using the equation, 关Ca2⫹兴 ⫽ K⬘d 关共R ⫺ Rmin兲/共Rmax ⫺ R兲兴共1/n兲,

(Eq. 1)

where Rmax and Rmin are the ratios obtained, respectively, in the absence of Ca2⫹ and at saturating Ca2⫹. K⬘d is the apparent dissociation constant and n is the Hill coefficient of the Ca2⫹ calibrations curves obtained in situ for each cameleon. For optimal representation, widefield fluorescence image stacks were deconvoluted after acquisition by treatment on a Silicon Graphics Octane work station using the Huygens 2 software (Bitplane AG, Zurich, Switzerland). For spectroscopic measurements, YC2 was extracted from stable transfectants by sonication. Membranes were removed by centrifugation (10,000 rpm, 10 min) and the supernatant diluted 1:8 before measuring the spectral response on a LS-5 fluorimeter (PerkinElmer Life Sciences). Image Analysis—The percentage of saturated pixels (Fig. 3) was determined image by image using a Metamorph routine, using a minimal size criteria of 5 contiguous pixels. A low-intensity threshold was used to define the cell-associated fluorescence signal (total pixel area) and a high-intensity threshold, corresponding to the average intensity of the total pixel area at saturating Ca2⫹ concentration, was applied to extract the saturated regions. To define ER regions close to mitochondria (Figs. 6 and 7), mitotracker red images were acquired just before and after agonist application. The location of mitochondria did not change significantly during this 2-min period. The red image was aligned to the yellow cameleon image using the nucleus boundaries, and used as a mask to define the mitochondria-associated ER region, which averaged 62% of the total ER area. To avoid the shading artifacts in the ratio images caused by the higher motility of the organelle edges, [Ca2⫹]ER was measured as F535 fluorescence. The F535 data were spatially averaged within the two regions and normalized to the fluorescence at the start of the experiment (F/F0). To compare ER regions located at similar distances from the cell border, the cell periphery was defined on the ER image and eroded by 5 and 10 pixels (1.3–2.6 ␮M). The nuclear area was excluded and the mitotracker image used to define mitochondria-rich ER regions. RESULTS

FIG. 2. Ca2ⴙ calibration and pH dependence. a, in situ Ca2⫹ calibrations of HEK-293 cells stably expressing YC2 (●) and YC4ER. (E). Cells were incubated for several minutes with 10 ␮M ionomycin at the indicated calcium concentration, pH 7.4. For YC4ER calibration, the solution also contained 5 ␮g/ml digitonin. To mimic the conditions in the mitochondrial matrix, YC2 was also calibrated at pH 8.0 in the presence of 10 ␮M nigericin and 10 ␮M monensin (f). b, changes in EYFP emission fluorescence during Ca2⫹ and pH changes in HeLa cells expressing YC4ER (●) or YC2mit (E). Cells were stimulated sequentially with 50 ␮M histamine (Hist), 1 ␮M thapsigargin (Tg), 1 ␮M ionomycin (iono), 1 ␮M of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), and 25 mM of the permeant weak base NH4Cl. The pH of the organelle was then equilibrated at the indicated pH values with 10 ␮M nigericin and 10 ␮M monensin. c, in situ Ca2⫹ calibrations of HeLa cells transiently expressing the mitochondrial probes YC3.1mit and YC4.1mit. Cells were equilibrated with 10 ␮M ionomycin, 10 ␮M nigericin, and 10 ␮M monensin at the indicated calcium concentration, pH 8.0. expression of the probes. [Ca2⫹] Measurements—Cells plated on 25-mm coverslips were superfused at 37 °C in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and vacuum outlet for solution changes. The method for dual-emission ratio imaging of [Ca2⫹] with the use of cameleons was derived from Ref. 43. Cameleon fluorescence from cells was imaged on a Axiovert S100 TV using a 100X, 1.3 NA oil-immersion objective (Carl Zeiss AG, Feldbach, Switzerland). Cells were excited by the 430 ⫾ 10 nm line from a monochromator (DeltaRam, Photon Technology International Inc., Monmouth Junction, NJ) through a 455DRLP dichroic mirror.

Targeting of Probes to Organelles—To measure Ca2⫹ variations in the cytoplasm and endoplasmic reticulum, we used two of the original yellow cameleons Ca2⫹ indicators: YC2, devoid of retention signal, and YC4ER, a low affinity indicator with a KDEL sequence and a calreticulin signal peptide (43). A series of new cameleons bearing the cytochrome oxidase complex VIII targeting sequence were used to measure Ca2⫹ in the mitochondria: YC2mit, a high affinity probe, as well as YC3.1mit and YC4.1mit, two mitochondrial probes of intermediate and low Ca2⫹ affinity, respectively. These two probes had a reduced pH dependence as they were based on the “improved” cameleons containing the V68L and Q69K mutations in the EYFP module (43, 44). Fig. 1 illustrates the subcellular distribution of the YC2, YC4ER, and YC2mit probes in transiently transfected HeLa cells. A three-dimensional rendering of wide-field images processed with a deconvolution algorithm is shown. As expected, the YC2 probe was uniformly distributed throughout the cytosol, with “holes” corresponding to the volume displaced by organelles (Fig. 1a). In contrast, the YC4ER probe produced a delicate reticular staining pattern characteristic of the ER (Fig. 1b). The YC2mit probe labeled discrete structures aligned along a largely interconnected network (Fig. 1c), consistent with the typical pattern of mitochondria in intact HeLa cells (19). These staining patterns were observed in most cells transfected at low efficiency (1–5%), but higher transfection effi-


29433

FIG. 3. [Ca2ⴙ]mit transients reported by the three mitochondrial probes. a, left: YC2mit fluorescence image illustrating the total measured area (white line) and the regions larger than 5 contiguous pixels that saturated during the histamine response (red). The saturation level was defined as the average intensity of the total pixel area at saturating [Ca2⫹], as described under “Experimental Procedures.” Middle, changes in ratio fluorescence during stimulation with 50 ␮M histamine and subsequent equilibration at 10 mM and 1 nM [Ca2⫹]. Right, time course of the pixel saturation during histamine application and subsequent equilibration at saturating [Ca2⫹]. The YC2mit probe was saturated in ⬃25% of mitochondria during the peak [Ca2⫹]mit response. b, similar experiment illustrating the response obtained with the YC3.1mit probe, which saturated in ⬃18% of mitochondria. A titration calibration was performed at the end of the experiments to estimate the peak [Ca2⫹]mit values. c, the YC4.1mit probe saturated in only ⬃3% of mitochondria and reported peak [Ca2⫹]mit levels around 100 ␮M. Traces are representative of 15, 8, and 14 experiments. Size bar: 5 ␮m.

ciency was avoided as it caused significant mistargeting. To confirm that these staining patterns corresponded to the ER and mitochondria, the localization of the probes was verified by co-localization with specific markers. The YC4ER probe largely co-localized with the ER marker protein calreticulin (Fig. 1b), while YC3.1mit co-localized with the vital dye mitotracker red, as observed by confocal microscopy (Fig. 1c). This confirmed that the two probes specifically labeled the ER and the mitochondria, respectively. Calibration and pH Dependence—The cameleons probes were calibrated in situ by incubating HEK-293 cells stably expressing YC2 or YC4ER for several minutes in heavily Ca2⫹buffered solutions containing 10 ␮M ionomycin. To ensure a good Ca2⫹ equilibration across the ER membrane, 5 ␮g/ml digitonin was included for YC4ER calibration. As shown in Fig. 2a, the YC2 calibration curve was monophasic, with an apparent dissociation constant (K⬘d) of 1.24 ␮M and a Hill coefficient (n) of 0.79. The small shoulder reflecting Ca2⫹ binding to the high affinity site of the calmodulin-M13 hybrid protein was not resolved in our in situ calibration curve, which was slightly shifted to the left compared with the curve obtained in vitro (43). In contrast, the YC4ER curve was clearly biphasic, with

K⬘d of 39 nM and 292 ␮M, and n of 0.57 and 0.60, consistent with the large shift in the low-affinity component produced by the E31Q mutation. The YC2 probe had a similar Ca2⫹ dependence when calibrated at pH 8.0 (squares), indicating that the probe behaved adequately in the pH range 7.0 – 8.0. To confirm that the pH dependence of the YC probes did not interfere with Ca2⫹ measurements, we monitored the pH of the ER and mitochondria. By directly exciting the pH-sensitive EYFP module (excitation/emission: 480/535 nm), Ca2⫹-dependent FRET is bypassed and the probe reports only pH changes. As shown in Fig. 2b, no pH changes were observed within the ER and mitochondria during histamine or thapsigargin stimulation. Ionomycin caused a slight decrease as Ca2⫹ was exchanged for H⫹ across the organelle membrane. In contrast, large changes in EYFP fluorescence were observed with the protonophore carbonylcyanide m-chlorophenylhydrazone or the permeant weak base NH4Cl. A pH titration curve in the presence of nigericin and monensin confirmed that the probe adequately reported the alkaline pH of mitochondria and the near-neutral pH of the ER. Thus, consistent with previous studies (48, 49), the pH of the ER and mitochondria is stable during Ca2⫹ transients, implying that, despite their pH de-

29434


TABLE I Histamine [Ca2⫹]mit transients reported by the three mitochondrial probes For each probe, the spatially averaged mitochondrial Ca2⫹ response (peak [Ca2⫹]mit) was calculated using the apparent Ca2⫹ affinity determined in situ (K⬘d), and the percentage of saturated pixels determined as described under “Experimental Procedures.” Data are mean ⫾ S.E. K⬘d

YC2mit YC3.1mit YC4.1mit

1.26 3.98 104

Peak [Ca2⫹]mit

Saturated pixels

␮M

%

3.19 ⫾ 0.39 49.4 ⫾ 7.4 106 ⫾ 5.0

24.8 ⫾ 3.9 17.5 ⫾ 3.1 3.19 ⫾ 0.8

n

15 8 14

pendence, the YC probes can be used to measure Ca2⫹ changes within these organelles. The pH independent probes targeted to the mitochondria were also calibrated in situ, using transiently transfected HeLa cells (Fig. 2c). The low-affinity mitochondrial probe YC4.1mit retained its biphasic Ca2⫹ dependence, with K⬘d of 105 nM and 104 ␮M, and n of 0.81 and 0.62. The YC3.1mit probe had intermediate Ca2⫹ affinity, with a K⬘d of 3.98 ␮M and n of 0.67, as expected from the E104Q mutation which completely eliminates the high-affinity Ca2⫹-binding site. Thus, the Ca2⫹ dependence of the probes was not affected by the environment of the mitochondrial matrix. Together with YC2mit, whose behavior was similar to the YC2 probe (not shown), the new mitochondrial probes allowed [Ca2⫹]mit measurements over a wide range of Ca2⫹ concentrations. [Ca2⫹]mit Transients Reach Submillimolar Levels in a Fraction of Mitochondria—Fig. 3 illustrate the [Ca2⫹]mit responses reported by the three mitochondrial probes. Individual organelles were difficult to resolve due to the high density and the motility of mitochondria, and the shrinkage of the organelle during equilibration at high [Ca2⫹] prevented a pixel-by-pixel calibration. In these conditions, only the spatially averaged signal could be adequately calibrated. Using the apparent dissociation constants obtained in situ, the resting [Ca2⫹]mit values measured with the high affinity YC2mit probe (Kd ⬃1.2 ␮M) averaged 188 ⫾ 25 nM (n ⫽ 15, range 48 – 445) and increased to 3.34 ⫾ 0.36 ␮M upon stimulation with histamine. This averaged value was clearly an underestimate, as ⬃25% of the signal was saturated during the peak histamine response (defined as objects larger than 5 contiguous pixels exceeding the averaged Rmax, Fig. 3a, right panel). Because the YC2mit probe saturates above 100 ␮M (Fig. 2), this suggested that the peak [Ca2⫹]mit values were higher than 100 ␮M in a fraction of mitochondria. A similar behavior was observed with the YC3.1mit probe (Kd ⬃4 ␮M), which saturates above 300 ␮M (Fig. 2). Although the spatially averaged values were substantially higher (Table I), a fraction of the signal was still saturated during the histamine response and a subsequent Ca2⫹ titration to 100 ␮M and 1 mM caused only a slight increase in ratio (Fig. 3b, middle panel). To determine unambiguously the peak [Ca2⫹]mit levels in these mitochondria, cells were transfected with the low affinity probe YC4.1mit (Kd ⬃105 ␮M), which responds adequately in the range 10 ␮M to 3 mM (Fig. 2c). The peak [Ca2⫹]mit reported by the YC4.1mit probe averaged 106 ⫾ 5 ␮M, and a further increase was observed when cells were equilibrated with 1 mM free [Ca2⫹] (Fig. 3c and Table I). The peak [Ca2⫹]mit value now comprised almost all responding mitochondria, as only ⬃3% of the YC4.1mit signal saturated during the histamine response. Thus, the response of mitochondria was quite heterogenous, the [Ca2⫹]mit transients reaching millimolar levels in some mitochondria, which are likely to be very close to the sites of Ca2⫹ release. ER Refilling and Heterogeneity during Stimulation with IP3generating Agonists—Similar to mitochondria, the ER was also

quite motile and exhibited some “ruffling” that prevented a pixel-by-pixel calibration, but in this case the stability of the overall structure allowed to compare distinct ER regions. Fig. 4 illustrates the [Ca2⫹]ER responses elicited by histamine (Fig. 4a, linked video: YC4ER-hist.mov) and by the SERCA ATPase inhibitor thapsigargin (Fig. 4c, linked video: YC4ER-TG.mov). The resting [Ca2⫹]ER levels averaged 371 ⫾ 21 ␮M in the absence of external Ca2⫹ (n ⫽ 35, range 187–585) and decreased rapidly to 133 ⫾ 14 ␮M upon stimulation with histamine (Fig. 4a, linked video: YC4ER-hist.mov). This decrease sometimes displayed an oscillatory pattern (not shown), suggesting that the ER refilled during stimulation. Accordingly, [Ca2⫹]ER rapidly returned to prestimulatory levels upon Ca2⫹ readdition. In contrast, TG induced a slower but significantly larger [Ca2⫹]ER decrease than histamine (Fig. 4c, linked video: YC4ER-TG.mov) the final levels averaging 59 ⫾ 5 ␮M (Fig. 4d, n ⫽ 8). Subsequent readdition of Ca2⫹ had no effects, confirming that SERCAs had been fully inhibited and that this value reflected the fully depleted [Ca2⫹]ER levels. Thus, during stimulation with IP3-generating agonists, cells maintain [Ca2⫹]ER well above depleted levels, indicating that substantial ER refilling occur even in the absence of external Ca2⫹. Subcellular analysis of the ER responses revealed that distinct ER regions had similar resting [Ca2⫹]ER values, and that thapsigargin released Ca2⫹ with similar kinetics throughout the ER network (Fig. 4d). This suggests that Ca2⫹ equilibrates freely within the ER lumen, both at rest and during the slow depletion induced by TG. In contrast, stimulation with histamine produced a more heterogeneous [Ca2⫹]ER response, the ER depleting faster in regions closer to the plasma membrane (Fig. 4b). This heterogeneity was not due to a higher motility of the organelle during the histamine response, as verified by time-lapse recordings (see movies). Rather, it might reflect the increased activity of plasma-membrane ATPases upon stimulation with IP3 generating agonists. Alternatively, the heterogeneity might reflect the distinct mitochondria density of the central and perinuclear ER regions, which could differentially modulate the kinetics of IP3-induced Ca2⫹ release. Ca2⫹ Cycles between Mitochondria and the ER—To assess the functional role of the mitochondria/ER interactions, we studied the effect of mitochondrial inhibitors on the Ca2⫹ signals measured in the different cell compartments (Fig. 5). Histamine induced oscillations in [Ca2⫹]cyt that were readily detected by mitochondria (Fig. 5, a and b, left panels). These oscillations originated from the ER as they occurred in the absence of external Ca2⫹, and could be reproduced repeatedly when cells were allowed to refill between consecutive histamine applications (Fig. 5a). This approach allowed us to study the contribution of mitochondria without compounding effects due to store-operated Ca2⫹ influx, which has been shown to be strongly inhibited by mitochondrial inhibitors (50). To collapse the mitochondrial membrane potential while minimizing ATP depletion, we used a combination of rotenone, an inhibitor of mitochondrial respiratory complex I, and oligomycin, to prevent ATP consumption by the reverse function of the mitochondrial H⫹-ATPase (51, 52). As expected, oligomycin/rotenone fully inhibited Ca2⫹ uptake by mitochondria, the peak [Ca2⫹]mit becoming almost undetectable even with the high-affinity YC2mit probe (Fig. 5b, peak [Ca2⫹]mit ⫽ 2.92 ⫾ 0.43 ␮M versus 0.48 ⫾ 0.11 ␮M, n ⫽ 7). More Ca2⫹ was released into the cytosol when mitochondrial Ca2⫹ uptake was blocked, the averaged [Ca2⫹]cyt levels measured with the cytosolic YC2 probe increasing by 20 ⫾ 8% (n ⫽ 6, p ⬍ 0.05). Interestingly, the peak response was not significantly affected, but [Ca2⫹]cyt failed to return to basal levels between oscillations (Fig. 5a, right panel), suggesting that


29435

FIG. 4. Heterogeneity of [Ca2ⴙ]ER responses to IP3 generating agonists. a, intensity modulated YC4ER ratio image (linked video: YC4ER-hist.mov) illustrating the total cell area (white lines) and the measured regions of interest. b, time course of the [Ca2⫹]ER response to histamine in Ca2⫹-free medium, followed by wash of the agonist and Ca2⫹ readdition. Regions far from the nucleus (dotted lines, R2, R5) released more Ca2⫹ than regions close to nucleus (dashed lines, R1, R3, R4). The black line is the mean response of the entire cell. c, left: intensity modulated YC4ER ratio image (linked video: YC4ER-TG.mov) of cells stimulated with the SERCA ATPases inhibitor thapsigargin (Tg). d, time course of the Tg response. Tg induced a slower and more homogenous decrease in [Ca2⫹]ER.

Ca2⫹ clearance from the cytosol was affected by the lack of functional mitochondria. Parallel [Ca2⫹]ER measurements revealed that the lack of mitochondrial Ca2⫹ uptake was associated with an increased depletion of ER Ca2⫹ stores (Fig. 5c). Due to the small dynamic range of YC4ER, reproducible [Ca2⫹]ER responses could not be obtained by repetitive stimulation of the same cell. However, in independent experiments [Ca2⫹]ER levels decreased from 304 ⫾ 46 to 126 ⫾ 24 ␮M in control and from 299 ⫾ 43 to 69 ⫾ 15 ␮M in oligomycin/rotenone-treated cells, corresponding to a [Ca2⫹]ER decrease of 59 ⫾ 3 versus 75 ⫾ 5%, respectively (mean ⫾ S.E., n ⫽ 6, p ⫽ 0.03, unpaired t test). Interestingly, the initial drop in [Ca2⫹]ER induced by the agonist was preserved, but [Ca2⫹]ER continued to decrease in the presence of the mitochondrial inhibitors during the stimulation (Fig. 5c, right). This did not reflect a lack of Ca2⫹ pumping ability of the ER, as upon Ca2⫹ readdition [Ca2⫹]ER increased with similar kinetics regardless of the presence of the inhibitors (Fig. 5c, ␶1⁄2 ⫽ 29 ⫾ 7 s versus 38 ⫾ 10 s for oligomycin/rotenone). This indicates that the SERCA ATPases were still fully functional and that the increased ER depletion was not due to a decrease in ATP levels. To test whether the larger ER depletion reflected the lack of Ca2⫹ returning from mitochondria, we used CGP 37157, a blocker of the mitochondrial Na⫹/Ca2⫹ exchanger. CGP 37157 caused a marked prolongation of the [Ca2⫹]mit signal as Ca2⫹ remained trapped in the mitochondria (Fig. 5d). Although this effect is expected to increase the production of oxidative ATP, the decrease in [Ca2⫹]ER was larger and more

sustained in the presence of CGP 37157 (Fig. 5e). In nine independent experiments, [Ca2⫹]ER decreased from 381 ⫾ 25 to 175 ⫾ 19 ␮M in control and from 314 ⫾ 26 to 90 ⫾ 10 ␮M in CGP-treated cells, corresponding to a 53 ⫾ 5 versus 71 ⫾ 2% decrease in [Ca2⫹]ER, respectively (p ⫽ 0.013, unpaired t test). The increased ER depletion observed in the presence of CGP 37157 thus suggests that part of the Ca2⫹ captured by mitochondria is normally re-used for ER refilling. Mitochondria Define Two Functional Ca2⫹ Stores in the ER—To determine whether mitochondria indeed sustain the refilling of the ER, the kinetics of Ca2⫹ release were determined in ER regions containing or lacking mitochondria (Fig. 6). Cells were co-labeled with mitotracker red to locate mitochondria (Fig. 6b), and [Ca2⫹]ER measured within overlapping or non-overlapping ER regions, which comprised 61.6 ⫾ 1.6 and 38.4 ⫾ 1.6% of the total YC4ER staining, respectively. To avoid artifacts caused by the organelle motility, [Ca2⫹]ER was measured as the ratio of F535 fluorescence normalized to the fluorescence at the start of the experiment (F/F0). Consistent with the results of Fig. 4 upon stimulation with histamine [Ca2⫹]ER decreased more slowly in the central ER regions that contained mitochondria (Fig. 6c, gray dots). Statistical analysis confirmed that the [Ca2⫹]ER levels measured at the end of the histamine stimulation were significantly lower in ER regions lacking mitochondria (Table II). The difference persisted when ER regions located between 1.3 and 2.6 ␮M from the cell border were compared (0.982 ⫾ 0.005 versus 0.956 ⫾ 0.014, n ⫽ 7, p ⬍ 0.05), indicating that the faster depletion of the mitochondria-free

29436


FIG. 5. Effects of mitochondria inhibitors on Ca2ⴙ signals. Cells were stimulated with histamine in the absence of external calcium and incubated for 10 min with the mitochondrial inhibitors in calcium-containing medium between histamine applications. a-c, effect of 5 ␮g/ml oligomycin and 25 ␮M rotenone on the changes in [Ca2⫹]cyt, [Ca2⫹]mit, and [Ca2⫹]ER induced by histamine. The same cell was stimulated twice, except for [Ca2⫹]ER measurements. In c, 1 mM Ca2⫹ was added at the end of the histamine stimulation to illustrate the ER refilling kinetics. The mitochondrial inhibitors largely abolished the cytosolic Ca2⫹ oscillations and completely blocked mitochondrial Ca2⫹ uptake, but potentiated the decrease in [Ca2⫹]ER. d and e, effect of 10 ␮M CGP 37157 on the histamine-induced changes in [Ca2⫹]mit and [Ca2⫹]ER. The block of the mitochondrial Na⫹/Ca2⫹ exchanger prolonged the [Ca2⫹]mit signal and increased the ER depletion. Traces are representative of 6 –14 experiments.

compartment did not reflect its closer proximity to the plasma membrane. Consistent with a role of mitochondria in ER refilling, the differences disappeared in the presence of thapsigargin, the ER depletion being even slightly faster in ER regions containing mitochondria (Fig. 6, d-f, and Table II). Ca2⫹ release occurred with identical kinetics throughout the ER network in the presence of oligomycin/rotenone (Fig. 7, a-c, and Table II), confirming that the local refilling of neighboring ER regions required functional mitochondria. No differences were observed between the two ER regions in the presence of CGP 37157 (Fig. 7, d-f, and Table II), confirming that localized ER refilling depended on the supply of mitochondrial Ca2⫹, rather than on the local ATP levels. Taken together, these experiments indicate that mitochondria prevent the depletion of vicinal ER regions by returning Ca2⫹ back into the ER. The presence of active mitochondria thus defines two functionally distinct Ca2⫹ stores within the ER lumen. DISCUSSION

In this study, we used green fluorescent protein-based cameleon probes to measure Ca2⫹ changes within the cytosol, ER,

and mitochondria. These genetically encoded Ca2⫹ indicators offer several advantages over other approaches used to measure Ca2⫹ changes in organelles. Their bright fluorescence and molecular targeting allowed time-resolved imaging of Ca2⫹ signals in defined intracellular compartments. The tunable Ca2⫹ affinity of the ratiometric probes ensured quantitative measurements within a wide range of Ca2⫹ concentrations. Furthermore, the probes could be used in conjunction with red shifted dyes to study the interactions between organelles (Figs. 6 and 7). Among the disadvantages, we observed that the targeting efficiency depended on cameleon expression levels, and that the small dynamic range of the probes limited the precision of the measurements. A more significant drawback was the pH dependence of the first generation of cameleons, which precluded the use of protonophores and required independent determination of the pH of the organelle. The stable pH of the ER and the alkaline pH of the mitochondria, however, allowed us to use the pH-sensitive cameleons for Ca2⫹ measurements in these organelles. The resting [Ca2⫹]ER levels averaged 371 ␮M in HeLa cells,


29437

FIG. 6. Mitochondria define two functional Ca2ⴙ stores in the ER. a-c, YC4ER cells were co-labeled with mitotracker red to locate mitochondria, and [Ca2⫹]ER was measured in regions containing (white line) and lacking mitochondria (dashed gray line). The two regions had similar ratio values prior to the histamine stimulation (2.52 versus 2.62), and [Ca2⫹]ER was measured by following the decrease in F535 fluorescence (F/F0) to avoid artifacts in the ratio image caused by the motility of the organelle edges. The ER region containing mitochondria (open circles) had higher [Ca2⫹]ER levels during the histamine stimulation than the ER region lacking mitochondria (dashed gray circles). d-f, the same experiment was performed in the presence of thapsigargin to inhibit the refilling of the ER. The differences between ER regions disappeared when SERCA ATPases were inhibited. Data are representative of seven (a-c) and five (d-f) experiments. Size bar, 5 ␮m. TABLE II YC4ER responses in ER regions containing or lacking mitochondria The decrease in [Ca2⫹]ER (⌬F/F0) was measured in YC4ER-labeled regions that co-localized or not with the mitotracker red staining (% mito), as described under “Experimental Procedures.” Data are mean ⫾ S.E. p ⫽ paired Student’s t test. Pixel area (%) Mito poor

Control Tg Oligo/Rot CGP

Pixel area(%) 43.9 ⫾ 2.2 26.7 ⫾ 4.9 40.7 ⫾ 1.1 38.4 ⫾ 1.5

Mito rich

⌬F/F0 10.3 ⫾ 1.8 5.6 ⫾ 0.9 7.3 ⫾ 3.0 5.6 ⫾ 1.1

Pixel area(%) 56.1 ⫾ 2.2 73.3 ⫾ 4.9 59.3 ⫾ 1.1 61.6 ⫾ 1.5

⌬F/F0 4.4 ⫾ 0.7 6.8 ⫾ 0.2 9.7 ⫾ 2.1 6.6 ⫾ 2.1

n

p

7 5 4 7

0.003 0.24 0.13 0.72

FIG. 7. Mitochondria recycle Ca2ⴙ back to vicinal ER domains. Experiments were performed as described in the legend to Fig. 6, but in the presence of oligomycin/rotenone (a-c) or CGP 37157 (d-f). The differences between the two ER regions disappeared in the presence of the mitochondrial inhibitors. Data are representative of four (a-c) and seven (d-f) experiments. Size bar: 5 ␮m.

values that agree well with previous reports using aequorin or cameleons (39, 41, 43, 47, 53). Substantial ER refilling was observed during agonist stimulation even in the absence of extracellular Ca2⫹, consistent with a recent study using targeted cameleon (47). No Ca2⫹ gradients were observed within the ER at rest or during stimulation with thapsigargin (Fig. 4), suggesting that the ER behaves as a single continuous compartment when IP3 levels are low. In contrast, two functionally distinct ER compartments were observed during stimulation with IP3-generating agonists. During histamine stimulation, regions rich in mitochondria, located deep in the cytosol, had higher [Ca2⫹]ER levels that regions poor in mitochondria, located at the periphery of the cell (Fig. 6). The difference persisted in ER regions located at similar distance from the cell

border, indicating that it did not reflect Ca2⫹ extrusion by the plasma-membrane ATPases. Instead, the two functional ER subdomains reflected the differential activity of SERCA ATPases, as the ER inhomogeneity disappeared in the presence of thapsigargin (Fig. 6b). A large part of the repumped Ca2⫹ originated from mitochondria, as the depletion of ER Ca2⫹ stores increased by 18% when mitochondrial Ca2⫹ efflux was blocked with CGP 37157 (Fig. 5). Previous studies have shown that mitochondria are very close to the ER in HeLa cells (19), and that Ca2⫹ signal transmission between these organelles is quasisynaptic (54). Consistent with these observations, we observed that [Ca2⫹]mit transients reached submillimolar values in a fraction of mitochondria (Fig. 3), which must be very close to the sites of Ca2⫹

29438


release. These high levels were not detected by the high affinity YC2mit probe, which was near saturation within this range of Ca2⫹ concentrations, but were readily detected by the lowaffinity YC4.1mit probe, which reported an average peak [Ca2⫹]mit around 100 ␮M (Fig. 3). This finding is consistent with recent imaging data obtained in HeLa cells with a permutated green fluorescent protein engineered to sense Ca2⫹ (55), suggesting that earlier reports using high-affinity fluorescent dyes or aequorin might have underestimated the peak [Ca2⫹]mit response. Higher, millimolar values were reported in chromaffin cells using a mutated aequorin of reduced Ca2⫹ affinity (36), possibly reflecting the close proximity of these mitochondria to both the plasma membrane and Ca2⫹ release channels (36). In HeLa cells, mitochondria are located far from the plasma membrane. The high [Ca2⫹]mit levels observed in HeLa cells thus indicate that mitochondria take up a significant portion of the Ca2⫹ released by the ER. Some of this Ca2⫹ is then re-used for ER refilling, suggesting that Ca2⫹ cycles back and forth between the ER and mitochondria during Ca2⫹ oscillations. Mitochondria might increase ER refilling by providing a local source of either Ca2⫹ or ATP, thereby enhancing the activity of SERCA ATPases. The Ca2⫹ effect appear predominant, as block of mitochondrial Ca2⫹ efflux by CGP 37157, which should increase the production of ATP by mitochondria, enhanced the depletion of the ER and led to the disappearance of the mitochondria-associated compartment. Furthermore, the ER refilled with similar kinetics regardless of the presence of mitochondria, indicating that the mitochondrial ATP did not contribute significantly to the activity of the SERCA ATPases. Accordingly, the ER refilling kinetics were not affected by oligomycin/rotenone or CGP 37157, consistent with earlier observations indicating that ATP generation is mainly glycolytic in HeLa cells (56). Thus, the predominant effect of mitochondria is to provide the local source of Ca2⫹ for refilling the ER. The close proximity of mitochondria from the sites of Ca2⫹ release ensures that a large part of the escaped Ca2⫹ is returned back into the ER. In previous studies using trapped fluorescent dyes or BAPTA-loaded cells, mitochondria have been shown to increase, rather than prevent, the depletion of the ER (50, 53). This might reflect the effect of Ca2⫹ buffers on the ER/mitochondria interactions, as trapped fluorescent dyes have been shown to accumulate not only into the ER lumen, but also in the mitochondrial matrix (57). Because the peak [Ca2⫹]mit are much larger than previously thought (Fig. 3), low-affinity dyes such as Mag-fura can be significantly affected by the [Ca2⫹]mit changes. Accordingly, both increase and decreases in [Ca2⫹] have been reported during agonist stimulation using Mag-fura, depending on the relative contribution of mitochondria to the Mag-fura signal (57). The decreased [Ca2⫹] release observed with Mag-fura in the presence of mitochondrial inhibitors thus likely reflects the decreased [Ca2⫹]mit signal, rather than the true [Ca2⫹]ER response (50). In BAPTA-loaded cells, on the other hand, accumulation of the Ca2⫹ chelator might trap Ca2⫹ into the mitochondrial matrix, increasing the ability of mitochondria to buffer [Ca2⫹] but minimizing their contribution in the ER refilling process. Consistent with the ability of mitochondria to increase local Ca2⫹ buffering, we observed that mitochondria slightly increased the depletion of vicinal ER regions when refilling was blocked by thapsigargin (Fig. 6b). The local cycle of Ca2⫹ between a portion of the ER and its neighboring mitochondria generates two functionally distinct Ca2⫹ stores within the ER network. The two stores are not structurally distinct ER pools with different Ca2⫹ transport characteristics, because they behaved similarly in the presence of thapsigargin or when mitochondrial Ca2⫹ cycling was inhib-

ited (Figs. 6 and 7). Instead, they reflected the imbalance between the local Ca2⫹ refilling in ER regions bearing mitochondria and the Ca2⫹ drag occurring in remaining ER regions. Recent studies indicate that the ER is a lumenally continuous organelle, and that Ca2⫹ changes induced by local uncaging propagate over several micrometers (58). Accordingly, we found that [Ca2⫹]ER changes were homogenous within the ER lumen in the absence of functional mitochondria. In contrast, the presence of functional mitochondria allowed neighboring ER regions to maintain higher [Ca2⫹]ER levels. Although Ca2⫹ diffusion within the ER lumen would tend to dissipate these Ca2⫹ gradients, the high [Ca2⫹]mit levels detected in some mitochondria indicate that mitochondria might provide sufficient amounts of Ca2⫹ to achieve a local control of [Ca2⫹]ER levels in neighboring ER regions. The strategic location of mitochondria thus appears to be a key determinant of their ability to modulate Ca2⫹ signals. By preventing the depletion of a defined ER region, mitochondria can restrict Ca2⫹ signals to specific regions of the cell. Depletion of the peripheral ER region could be required for the activation of store-operated Ca2⫹ influx, while refilling of the central ER region allow generating Ca2⫹ oscillations near the nucleus. The depletion of the central ER region was indeed associated with an altered cytosolic Ca2⫹ signal as, in the presence of mitochondria inhibitors, [Ca2⫹]cyt failed to return to basal levels between oscillations (Fig. 4). The cycling of calcium between the ER and mitochondria thus not only controls the filling state of the ER, but also the spatio-temporal pattern of the cytosolic Ca2⫹ signal. Acknowledgments—We thank Cyril Castelbou for dedicated technical assistance, Drs. R. Y. Tsien and A. Miyawaki for providing the cameleon constructs, Dr. Uta Schmidt for expert advice, and Drs L. Bernheim, M. Mu¨hlethaler, and W. Graier for critical reading of the manuscript. REFERENCES 1. Clapham, D. E. (1995) Cell 80, 259 –268 2. Berridge, M. J., Bootman, M. D., and Lipp, P. (1998) Nature 395, 645– 648 3. Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., and Lederer, W. J. (1995) Science 270, 633– 637 4. Bito, H., Deisseroth, K., and Tsien, R. W. (1996) Cell 87, 1203–1214 5. Berridge, M., Lipp, P., and Bootman, M. (1999) Curr. Biol. 9, R157–159 6. Neher, E. (1998) Neuron 20, 389 –399 7. Berridge, M. J. (1993) Nature 361, 315–325 8. De Koninck, P., and Schulman, H. (1998) Science 279, 227–230 9. Tse, A., Tse, F. W., Almers, W., and Hille, B. (1993) Science 260, 82– 84 10. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392, 933–936 11. Li, W., Llopis, J., Whitney, M., Zlokarnik, G., and Tsien, R. Y. (1998) Nature 392, 936 –941 12. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 415– 424 13. Rizzuto, R., Bastianutto, C., Brini, M., Murgia, M., and Pozzan, T. (1994) J. Cell Biol. 126, 1183–1194 14. Babcock, D. F., and Hille, B. (1998) Curr. Opin. Neurobiol. 8, 398 – 404 15. Duchen, M. R. (1999) J. Physiol. 516, 1–17 16. Rutter, G. A., and Rizzuto, R. (2000) Trends Biochem. Sci. 25, 215–221 17. Tinel, H., Cancela, J. M., Mogami, H., Gerasimenko, J. V., Gerasimenko, O. V., Tepikin, A. V., and Petersen, O. H. (1999) EMBO J. 18, 4999 –5008 18. Simpson, P. B., Mehotra, S., Lange, G. D., and Russell, J. T. (1997) J. Biol. Chem. 272, 22654 –22661 19. Rizzuto, R., Pinton, P., Carrington, W., Fay, F. S., Fogarty, K. E., Lifshitz, L. M., Tuft, R. A., and Pozzan, T. (1998) Science 280, 1763–1766 20. Hoth, M., Fanger, C. M., and Lewis, R. S. (1997) J. Cell Biol. 137, 633– 648 21. Babcock, D. F., Herrington, J., Goodwin, P. C., Park, Y. B., and Hille, B. (1997) J. Cell Biol. 136, 833– 844 22. Hajnoczky, G., Hager, R., and Thomas, A. P. (1999) J. Biol. Chem. 274, 14157–14162 23. Jouaville, L. S., Ichas, F., Holmuhamedov, E. L., Camacho, P., and Lechleiter, J. D. (1995) Nature 377, 438 – 441 24. Simpson, P. B., and Russell, J. T. (1996) J. Biol. Chem. 271, 33493–33501 25. Boitier, E., Rea, R., and Duchen, M. R. (1999) J. Cell Biol. 145, 795– 808 26. Kaftan, E. J., Xu, T., Abercrombie, R. F., and Hille, B. (2000) J. Biol. Chem. 275, 25465–25470 27. Hofer, A. M., and Machen, T. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2598 –2602 28. Hirose, K., and Iino, M. (1994) Nature 372, 791–794 29. Tse, A., Tse, F. W., and Hille, B. (1994) J. Physiol. 477, 511–525 30. Golovina, V. A., and Blaustein, M. P. (1997) Science 275, 1643–1648 31. Hofer, A. M., Fasolato, C., and Pozzan, T. (1998) J. Cell Biol. 140, 325–334 32. Rizzuto, R., Brini, M., and Pozzan, T. (1993) Cytotechnology 11, S44 – 46

Calcium Cycling between the ER and Mitochondria 33. Rizzuto, R., Brini, M., and Pozzan, T. (1994) Methods Cell Biol. 40, 339 –358 34. Rizzuto, R., Simpson, A. W., Brini, M., and Pozzan, T. (1992) Nature 358, 325–327 35. Rutter, G. A., Burnett, P., Rizzuto, R., Brini, M., Murgia, M., Pozzan, T., Tavare, J. M., and Denton, R. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5489 –5494 36. Montero, M., Alonso, M. T., Carnicero, E., Cuchillo-Ibanez, I., Albillos, A., Garcia, A. G., Garcia-Sancho, J., and Alvarez, J. (2000) Nat. Cell Biol. 2, 57– 61 37. Montero, M., Brini, M., Marsault, R., Alvarez, J., Sitia, R., Pozzan, T., and Rizzuto, R. (1995) EMBO J. 14, 5467–5475 38. Button, D., and Eidsath, A. (1996) Mol. Biol. Cell 7, 419 – 434 39. Barrero, M. J., Montero, M., and Alvarez, J. (1997) J. Biol. Chem. 272, 27694 –27699 40. Alonso, M. T., Barrero, M. J., Michelena, P., Carnicero, E., Cuchillo, I., Garcia, A. G., Garcia-Sancho, J., Montero, M., and Alvarez, J. (1999) J. Cell Biol. 144, 241–254 41. Pinton, P., Ferrari, D., Magalhaes, P., Schulze-Osthoff, K., Di Virgilio, F., Pozzan, T., and Rizzuto, R. (2000) J. Cell Biol. 148, 857– 862 42. Pinton, P., Pozzan, T., and Rizzuto, R. (1998) EMBO J. 17, 5298 –5308 43. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Nature 388, 882– 887 44. Miyawaki, A., Griesbeck, O., Heim, R., and Tsien, R. Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2135–2140

29439

45. Fan, G. Y., Fujisaki, H., Miyawaki, A., Tsay, R. K., Tsien, R. Y., and Ellisman, M. H. (1999) Biophys. J. 76, 2412–2420 46. Emmanouilidou, E., Teschemacher, A. G., Pouli, A. E., Nicholls, L. I., Seward, E. P., and Rutter, G. A. (1999) Curr. Biol. 9, 915–918 47. Yu, R., and Hinkle, P. M. (2000) J. Biol. Chem. 275, 23648 –23653 48. Kim, J. H., Johannes, L., Goud, B., Antony, C., Lingwood, C. A., Daneman, R., and Grinstein, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2997–3002 49. Wu, M. M., Llopis, J., Adams, S., McCaffery, J. M., Kulomaa, M. S., Machen, T. E., Moore, H. P., and Tsien, R. Y. (2000) Chem. Biol. 7, 197–209 50. Landolfi, B., Curci, S., Debellis, L., Pozzan, T., and Hofer, A. M. (1998) J. Cell Biol. 142, 1235–1243 51. Mohr, F. C., and Fewtrell, C. (1990) Am. J. Physiol. 258, C217–226 52. Fulceri, R., Bellomo, G., Mirabelli, F., Gamberucci, A., and Benedetti, A. (1991) Cell Calcium 12, 431– 439 53. Montero, M., Barrero, M. J., and Alvarez, J. (1997) FASEB J. 11, 881– 885 54. Csordas, G., Thomas, A. P., and Hajnoczky, G. (1999) EMBO J. 18, 96 –108 55. Nagai, T., Sawano, A., Park, E. S., and Miyawaki, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3197–3202 56. Jouaville, L. S., Pinton, P., Bastianutto, C., Rutter, G. A., and Rizzuto, R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13807–13812 57. Gurney, A. M., Drummond, R. M., and Fay, F. S. (2000) Cell Calcium 27, 339 –351 58. Park, M. K., Petersen, O. H., and Tepikin, A. V. (2000) EMBO J. 19, 5729 –5739