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Address correspondence to Rosario Rizzuto, Dept. Exp. Diagn. Med. Sect. Gen. Pathol., Via Borsari 46, I-44100 Ferrara, Italy. Tel.: 39-0532-291361. Fax: 39-0532-247278. E-mail: r.rizzuto{at}unife.it
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Abstract |
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Key Words: organelle; calcium; apoptosis; signal transduction; porin
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Introduction |
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Despite the wide acceptance of the phenomenon of localized Ca2+ signaling between the ER and mitochondria, no molecular information is yet available on this important topic, since neither the mitochondrial Ca2+ transporters nor the structural proteins involved in the formation of the close contacts between the two organelles have been identified. In this work, we have investigated the role of the voltage-dependent anion channel (VDAC) (Colombini, 1979; Mannella, 1998) of the outer mitochondrial membrane in modulating the Ca2+ response of the organelle. For this purpose, we have expressed a recombinant VDACGFP fusion protein and analyzed Ca2+ homeostasis at the subcellular level with recombinant Ca2+-sensitive probes. By this means, we observed that VDAC is a key determinant of Ca2+ permeability at the ERmitochondria contacts and thus is responsible for exposing the uptake systems of the inner mitochondrial membrane to the large [Ca2+] gradients needed for rapidly accumulating Ca2+ in the organelle upon cell stimulation.
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Results |
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The effect of VDAC overexpression on ER Ca2+ handling
We then tested whether the enhancement of mitochondrial Ca2+ uptake could be explained by an effect on the state of filling of the intracellular Ca2+ stores and/or the kinetics of Ca2+ release, i.e., whether the effect on mitochondria is secondary to a global alteration in Ca2+ signaling. For this purpose, the [Ca2+] of the ER lumen ([Ca2+]er) was measured with a specifically targeted aequorin chimera (erAEQmut) (Barrero et al., 1997) (Fig. 4)
. No significant difference could be appreciated between VDAC-overexpressing and control cells in the steady state [Ca2+]er (480 ± 32 µM in VDAC-overexpressing cells versus 472 ± 33 µM in controls, n = 20, P > 0.05), indicating that VDAC does not affect the state of filling of the Ca2+ stores. Upon stimulation with histamine, a rapid drop in [Ca2+]er was observed followed by a slower virtually complete release of stored Ca2+ (Fig. 4 A). The kinetics and amplitude of [Ca2+]er decrease were very similar in VDAC-overexpressing and control cells, indicating that the effect of VDAC on mitochondrial Ca2+ homeostasis does not depend on the modification of ER Ca2+ storage or release. Given that a minor fraction (10%) of transfected VDAC was detected in the microsomal fraction (thus also in the ER), we decided to directly demonstrate that it does not increase the Ca2+ leak from the ER (thus supporting the indirect evidence provided by the lack of effect on the steady state [Ca2+]er). For this purpose, we evaluated the rate of Ca2+ discharge from the ER upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in VDAC-overexpressing and control cells (Fig. 4 B). Where indicated, the cells were treated with the SERCA blocker 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ; 30 µM). This caused rapid emptying of ER Ca2+ with no difference in kinetics and amplitude between VDAC-overexpressing and control cells.
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VDAC increases the Ca2+ permeability of ERmitochondria contact sites
The two remaining explanations for the effects of VDAC are that either it generates an increase of outer membrane permeability at the ERmitochondria contacts, or, alternatively, it regulates the formation of the contacts themselves. To discriminate between these possibilities (schematically shown in Fig. 6
A), we took advantage of an inherent property of aequorin, i.e., its irreversible reaction and thus progressive consumption during the experiment.
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To further investigate the spatial relationship between ER and mitochondria, we used the immunocytochemistry approach. Briefly, we transfected HeLa cells with mtBFP or mtBFP + VDACGFP (control and VDAC-overexpressing, respectively), fixed the samples, incubated them with an anticalreticulin (a typical ER marker) antibody, and then treated the cells with a secondary antibody conjugated with Cy5. In doing so, we were able to image the cells with three different wavelengths simultaneously (see Materials and methods). The resulting images were deconvolved and overlapped (Fig. 7
, AD and ad). We then calculated the percentage of mitochondrial surface (mtBFP) that colocalized with calreticulin (Cy5) (see Materials and methods). In both the conditions (control and VDAC-overexpressing cells), the average colocalization was 29% (control, 33 ± 13% n = 3 and VDAC-overexpressing cells, 25 ± 13%, n = 4, P > 0.05), suggesting that at least in our experimental condition the presence of VDAC did not increase the number of close contacts between ER and mitochondria. Similar results were obtained using an anti-SERCA 2 antibody to label the ER (unpublished data). We also checked the relationship between mitochondria and Golgi apparatus in control and VDAC-overexpressing cells using an antihuman golgin-97 to label the organelle. The average colocalization of mitochondrial surface with the Golgi was
0.4% (control, 0.4 ± 0.2% and VDAC-overexpressing cells, 0.4 ± 0.3%, n = 6, P > 0.05), indicating that also the spatial arrangement between Golgi and mitochondria was not altered by the overexpression of VDAC.
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High speed imaging of mitochondrial Ca2+ changes reveals a shorter delay of the mitochondrial Ca2+ response
To directly demonstrate the role of VDAC in allowing the diffusion of the Ca2+ microdomain from the mouth of the IP3 receptor to the mitochondrial Ca2+ uniporter, we performed high speed single cell imaging of cytosolic and mitochondrial Ca2+ changes. For this purpose, HeLa cells were transfected with a mitochondrially targeted CaMgaroo (a Ca2+-sensitive yellow variant of GFP) (Baird et al., 1999; Griesbeck et al., 2001) and loaded with Fura-2/AM and were analyzed with an imaging system based on a highly sensitive camera and fast alternation of excitation and emission wavelengths. As described in detail in Materials and methods, with this system a CaMgaroo image and a Fura-2/AM ratio image pair are acquired within 25 ms every 200 ms for 39.6 s. Fig. 8
shows the kinetics of [Ca2+]c and [Ca2+]m increase upon histamine stimulation in control and VDAC-overexpressing cells. It is apparent that the [Ca2+]c rise is followed by a delayed (460 ± 98 ms, n = 6) upstroke of [Ca2+]m as described previously (Drummond et al., 2000; Gerencser and Adam-Vizi, 2001). Interestingly, this delay is significantly (P < 0.05) shortened in VDAC-overexpressing cells (200 ± 49 ms, n = 6). Moreover, in VDAC-overexpressing cells the amplitude of the peak [Ca2+]m rise is increased compared with controls (F/F0 = 0.063 ± 0.005 versus 0.047 ± 0.007, respectively, n = 6, P < 0.05) in good agreement with the results obtained with the aequorin probe.
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We then investigated whether the higher sensitivity to apoptosis can be attributed to the enhancement of mitochondrial Ca2+ uptake and the ensuing alteration of organelle morphology. To analyze mitochondrial morphology, HeLa cells were transfected with the fluorescent mitochondrial marker mtRFP and placed on the stage of the fluorescence microscope. Fig. 10 shows three images of control (mtRFP-transfected) and VDAC-overexpressing (VDACGFP- and mtRFP-cotransfected) cells acquired immediately before (time 0) and 30 and 60 min after the addition of the 10 µM ceramide. In both control and VDAC-overexpressing cells, the largely interconnected mitochondrial network can be recognized at time 0. In control cells, minimal changes can be appreciated after 30 min, which become more apparent after 60 min (rounding and breaking of the network). In VDAC-overexpressing cells, mitochondrial alterations are obvious after 30 min, and a complete rupture of the three-dimensional network can be noticed after 60 min.
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Discussion |
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In this paper, we identified VDAC as a positive regulator of mitochondrial Ca2+ accumulation. This was the case in all the cell types investigated, from skeletal myotubes to stable cell lines such as HeLa, ECV, and L929 cells. This effect was not due to a generalized increase in the permeability of the outer membrane and/or an effect on the uptake systems of the inner membrane as deduced by (a) the measurement of mitochondrial Ca2+ uptake rates in permeabilized cells and (b) the lack of effect on the [Ca2+]m rise elicited by Ca2+ entry through plasmamembrane channels. The possibility that VDAC is involved in the formation of new ERmitochondria contacts is ruled out by the data obtained with high affinity aequorin, which reveals the highly responding domains of the mitochondrial network.
Overall, our results indicate that VDAC overexpression affects the Ca2+ permeability at the domains of the ERmitochondria cross-talk. This was directly demonstrated by the direct double imaging of mitochondrial and cytosolic [Ca2+] changes using a targeted Ca2+-sensitive GFP chimera and a fluorescent dye, respectively. This experiment confirms previous data, indicating a delay between the cytosolic and the mitochondrial Ca2+ rise (Drummond et al., 2000; Gerencser and Adam-Vizi, 2001) and shows that this delay is drastically shortened by VDAC overexpression. These data indicate that the transfer of the [Ca2+] microdomain generated at the mouth of the IP3 receptors across the outer membrane that allows the low affinity uniporter of the inner membrane to be exposed to high [Ca2+] and thus rapidly accumulate Ca2+ in the matrix is critically dependent on the VDAC repertoire. Thus, the permeability of the outer membrane at the ERmitochondria contacts is a kinetic bottleneck for the process of mitochondrial Ca2+ homeostasis. This could depend on various nonmutually exclusive possibilities: (a) the selective clustering of VDAC channels at the ERmitochondria contact sites supported by immunofluorescence data of this paper and previous immunoelectron microscopy experiments that demonstrated the clustering of VDAC and ryanodine receptors at sarcoplasmic reticulum/mitochondria contacts (Shoshan-Barmatz et al., 1996), (b) a different state of opening in these domains (different states of opening of VDAC channels, responsible for differences in anion permeability, were described by Rostovtseva and Colombini [1996]), or (c) the strict dependence of a highly transient phenomenon, such as the diffusion of a short lasting [Ca2+] microdomain on a very high permeability of the outer membrane.
Finally, the VDAC-dependent enhancement of mitochondrial Ca2+ responses potentiates the effect of apoptosis inducers, such as ceramide, in causing early alterations of organelle morphology. Interestingly, these result well correlate with those of Voehringer et al. (2000) who compared the gene expression patterns of two cells lines derived from a B cell lymphoma that differed in sensitivity to apoptosis. While antioxidant genes were highly expressed in the apoptosis-resistant cell, VDAC was shown to be strongly up-regulated upon irradiation in the apoptosis-sensitive cells. Similarly, Madesh and Hajnoczky (2001) demonstrated a key role of VDAC in activation of permeability transition pore and ensuing release of cytochrome c upon oxidative stress. Our results provide a mechanism by which the VDAC repertoire, by enhancing mitochondrial Ca2+ changes and Ca2+-dependent changes in organelle morphology, controls the decoding of organelle Ca2+ signals and in particular the activation of mitochondrial apoptotic events. Interestingly, in a recent paper Csordas et al. (2002) demonstrated that a proapoptotic protein, tcBid, potentiates the transfer of IP3-mediated Ca2+ signals to mitochondria. Thus, a resident ion channel, VDAC, and a proapoptotic member of the Bcl-2 family, tcBid, appear to be involved in tuning the mitochondrial Ca2+ signal and controlling its capacity to trigger cell death.
Much work still needs to be done for the molecular elucidation of mitochondrial Ca2+ homeostasis. The clarification of the important and complex role played in this process by VDAC, the most abundant channel of the outer membrane, provides a first molecular clue, thus on the one hand suggesting novel regulatory mechanisms in cellular calcium signaling and on the other identifying a potential pharmacological target in this process of major pathophysiological interest.
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Materials and methods |
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Aequorin measurements
Aequorin reconstitution, luminescence measurements, and calibration into [Ca2+] values were performed as described previously (Chiesa et al., 2001). In the experiments, the cells were perfused with KRB (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 20 mM NaHCO3, 2 mM L-glutamine, and 20 mM Hepes, pH 7.4, at 37°C) supplemented with either 1 mM CaCl2 or 100 µM EGTA (KRB/EGTA). In the experiments with permeabilized cells, a buffer mimicking the cytosolic ionic composition, (intracellular buffer [IB]) was employed: 140 mM KCl, 10 mM NaCl, 1 mM K3PO4, 5.5 mM glucose, 2 mM MgSO4, 1 mM ATP, 2 mM sodium succinate, and 20 mM Hepes (pH 7.05 at 37°C). IB was supplemented with either 50 µM EGTA (free [Ca2+] < 10-8 M) (IB/EGTA) or an EGTA (2 mM)-buffered [Ca2+] of 1 µM (IB/1 µM Ca2+), 2 µM (IB/2 µM Ca2+), or 5 µM (IB/5 µM Ca2+). HeLa cells were permeabilized by 1-min incubation with digitonin (added to IB/EGTA) before luminescence measurements. All of the results are expressed as means ± standard error, and Student's t test was used for the statistic.
Immunocytochemistry
HeLa cells (control and VDAC-overexpressing cells) were fixed in 2% paraformaldehyde for 20 min at RT. The samples were then treated for 30 min at RT with Triton 0.1% and ethanolamine 0.1 M (Sigma-Aldrich) (pH 8). The cells were incubated 2 h at RT with the anticalreticulin chicken polyclonal antibody (1:200 in BSA 3% in PBS, pH 8 [Affinity BioReagents, Inc.]) the monoclonal anti-SERCA2 ATPase (1:100; Affinity BioReagents, Inc.) or the monoclonal antihuman Glogin-97 (1:100; Molecular Probes). After three washes with PBS (10 min each), a 1-h incubation at RT with the donkey antichicken IgG (Cy5-conjugated, 1:200; Jackson ImmunoResearch Laboratories) or the donkey antimouse IgG (AlexaFluor 594, 1:200; Molecular Probes), and three washes with PBS, the samples were mounted on slides with 90% glycerol. Some other samples were incubated at 4°C overnight with the anti-Porin 31HL (VDAC) human mAb (1:100; Calbiochem). After three washes with PBS, the cells were incubated for 1 h at RT with the donkey antimouse IgG (AlexaFluor 594), extensively washed in PBS, and mounted with 90% glycerol. Some cells were treated only with the secondary antibodies and used to calculate values of background (due to unspecific binding) in the samples. Intensity values were calculated that eliminated 99% of the unspecific background in those cells. A threshold value for specific binding was established as the mean plus two SDs of the 99th percentile values from five cells.
Image acquisition and deconvolution
Myotubes or HeLa cells were placed in a Leyden chamber (Medical Systems Corp.) on the stage of an inverted Nikon microscope equipped with epifluorescence, piezoelectric motorization of the objective (Physik Instrumente, GmbH & Co.) and filter-wheels on the excitation and emission optical paths (Sutter Instrument Co.). Images were taken using a Nikon 60x 1.4 NA PlanAPO objective lens, a back-illuminated CCD camera (Princeton Instruments) and the Metamorph software (Universal Imaging Corp.). For computational deblurring, a stack of images was acquired through the depth of the cell and processed using the EPR software developed by the U. Mass Group (Carrington et al., 1995). For images of Figs. 1 and 10, the pixel size was 166 nm, and for Fig. 7 the pixel size was 83 nm.
Analysis of colocalization of fluorescence signals
The data analysis and visualization environment was used to visualize images in three dimensions, superimpose them, and determine the extent to which they coincided (Lifshitz et al., 1994). A custom computer program was used to first threshold each three-dimensional image in order to identify pixels belonging to mitochondria. For the images in Fig. 1, three fluorescence thresholds were chosen for each image: a low one to eliminate as much of the background fluorescence as possible while completely preserving the mitochondrial structures, a high one which completely eliminates the background and main structures begun to disappear, and a medium one by which images were processed that represented a value between the previous two. These thresholded images were then used to count total number of pixels (i.e., the total volume) occupied by the mitochondria and VDAC. Colocalization was calculated as the number of voxels (volume pixels) occupied by both signals (namely, VDACGFP and mtBFP) over all voxels occupied by the VDACGFP signal in thresholded images (Moore et al., 1993; Lifshitz et al., 1994), in other words the percentage of the overexpressed VDAC localized to mitochondria.
For the analysis of the spatial relationship among the ER, mitochondria, and VDAC, the three-dimensional images of mtBFP distribution were used to define the mitochondrial volume. The images of mtBFP, VDACGFP, anticalreticulin, and anti-VDAC were first thresholded to eliminate background (as described in Immunocytochemistry). The thresholded mtBFP images were used to mask the corresponding thresholded VDAC and calreticulin images sets in order to restrict the analysis of colocalization to only those voxels corresponding to the mitochondria. The percentage of mitochondrial surface containing either VDACGFP, anti-VDAC, or anticalreticulin colocalization was expressed as the number of voxels (volume pixels) occupied by either VDACGFP, anti-VDAC, or anticalreticulin over all voxels occupied by the mitochondrial signal mtBFP. The percentage of VDAC colocalized with calreticulin was expressed as the number of voxels occupied by both VDAC and calreticulin over all voxels occupied by the VDAC (all within the mtBFP mask).
Simultaneous measurement of mitochondrial and cytoplasmic [Ca2+]
To monitor [Ca2+] in the cytosol and in mitochondria simultaneously, transfected HeLa cells were loaded with 2 µM Fura-2/AM in KRB 30 min at 37°C and 10 min at RT. Cells were then washed in the same solution, and [Ca2+]c and [Ca2+]m changes were determined using a high speed, wide field digital imaging microscope (ZhuGe et al., 1999 contains a complete description of this system). Briefly, the system is based on a custom built inverted microscope. An objective (Nikon 40x, 1.3 NA) forms an image on a 128 x 128 pixel (pixel size 333 nm x 333 nm) frame transfer charge-coupled device camera. Three different laser shutters controlled both the exposure time and the alternation of the three different excitation wavelengths. The sequence of excitation wavelengths was 350 and 380 nm for Fura-2/AM and 514 nm for CaMgaroo, coupled through a 525 DCXR epifluorescence dichroic. Fluorescence emission was filtered by an HQ525 long pass filter (dichroic and filter; Chroma Technology). For each sample, first a through-focus image stack for CaMgaroo (514 nm excitation) was acquired before stimulation (9 planes, 0.5 µm apart). After this, a series of 66, three wavelength image sets were acquired exposing the cell 5 ms for each wavelengths with a delay between images of 5 ms, and the acquisition was repeat every 200 ms for a total of 39.6 s After 2.4 s (12 image sets) from the beginning of the recording, cells were challenged with a 5-s puff of 10 µM histamine. For each experiment, the three-dimensional CaMgaroo stack was deconvolved using the EPR station mentioned above, an intensity threshold was chosen, and the thresholded stack was projected to form a two-dimensional mask image. This mask was used to identify mitochondrial pixels in the subsequent time series. The Fura-2/AM ratio images, corresponding to the CaMgaroo images, were calculated as 350/380. Analysis of the resulting CaMgaroo and Fura-2/AM images were performed using custom designed software running on a Silicon Graphics workstation.
Isolation of subcellular fractions and Western blot analysis
HeLa cells were plated in 10-cm Petri dishes; VDAC-overexpressing cells were transfected with 40 µg VDACGFP. 36 h after transfection, cells were washed twice in PBS, scraped, centrifuged (1,000 rpm, 5 min, 25°C), and resuspended in 4 ml of buffer A (10 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1.5 mM CaCl2). All of the following steps were performed at 4°C. Cells were homogenized in Glas-Glas homogenier and 2 ml of buffer B (0.75 M sucrose, 60 mM Tris-HCl, pH 7.4, 3 mM EDTA, 3 mM EGTA, 3 mM MgCl2, 3 mM PMSF, protease inhibitor cocktail [P-8340; Sigma-Aldrich], 3mM DTT) were immediately added. Homogenates were centrifuged at 510 g for 10 min, and then the supernatants were recentrifuged at the same speed. Mitochondria were pelleted by centrifugation at 9,000 g for 10 min, resuspended in buffer C (0.25 M sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM MgCl2, 1 mM PMSF, protease inhibitor cocktail, 1 mM DTT), centrifuged again in order to eliminate microsomal and cytosolic contamination, and resuspended in 200 µl of buffer C. The postmitochondrial supernatants were centrifuged at 15,000 g for 30 min in order to remove lysosomal fraction. To separate cytosolic and microsomal fraction, the final supernatants were filtered through a 0.2 µm (Schleicher & Schuell GmbH) filter. Protein determination was performed according to the Bradford method. The proteins were separated by SDS-PAGE on 14% gel, and the amount of endogenous VDAC, VDACGFP, and Bax, and the marker proteins SERCA and ß-tubulin was estimated by Western blotting using rabbit anti-VDAC (1:5,000; a gift from Dr. V. De Pinto, University of Catania, Catania, Italy) and anti-Bax (1:10,000; Santa Cruz Biotechnology) polyclonal antibodies and mouse antiß-tubulin (1:5,000; Santa Cruz Biotechnology) and anti-SERCA2 (Affinity BioReagents) mAbs revealed with and antirabbit and mouse IgG HRP-labeled secondary antibodies (1:10,000; Santa Cruz Biotechnology), respectively, according to standard protocols. Proteins were visualized by ECL (Amersham Biosciences).
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Footnotes |
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Acknowledgments |
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We thank Telethon-Italy (grant nos. 1285 and GTF01011), the Italian Association for Cancer Research, the Human Frontier Science Program, the Italian University Ministry, the Italian Space Agency, the National Research Council, the National Institutes of Health (HL 61297 and GM 61981), and the National Science Foundation (DBI 9724611 and DIR 9200027) for financial support. E. Rapizzi, M.R. Wiechowski, and G. Vandecasteele are recipients of Telethon, the Federation of European Biochemical Societies, and European Molecular Biology Organization long term fellowships, respectively. This research has been supported by a Marie Curie Fellowship (contract no. HPMF-CT-2000-00644).
Submitted: 17 May 2002
Revised: 1 October 2002
Accepted: 1 October 2002
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