Stable Interactions between Mitochondria and Endoplasmic Reticulum Allow Rapid Accumulation of Calcium in a Subpopulation of Mitochondria
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m302301200
ISSN1083-351X
AutoresLuisa Filippin, Paulo Magalhães, Giulietta Di Benedetto, Matilde Colella, Tullio Pozzan,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoTo better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca2+-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum. To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca2+-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum. Calcium is arguably the most versatile player within the cell. This second messenger is directly or indirectly involved in a myriad of processes that span virtually all physiological aspects of a cell, including its birth, health, disease, and death. From a generic point of view, calcium was often seen to exert its action exclusively through changes in cytosolic free calcium concentration ([Ca2+]c). 1The abbreviations used are: [Ca2+]c, cytosolic free Ca2+ concentration; [Ca2+]m, mitochondrial matrix free Ca2+ concentration; cyt, mt, and nu, prefixes denoting cytosolic, mitochondrial, and nuclear, respectively; ER, endoplasmic reticulum; F, fluorescence intensity; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; InsP3, inositol 1,4,5-triphosphate; PericamR, ratiometric pericam; GFP, green fluorescent protein; KRB, Krebs-Ringer buffer; CaM, calmodulin.1The abbreviations used are: [Ca2+]c, cytosolic free Ca2+ concentration; [Ca2+]m, mitochondrial matrix free Ca2+ concentration; cyt, mt, and nu, prefixes denoting cytosolic, mitochondrial, and nuclear, respectively; ER, endoplasmic reticulum; F, fluorescence intensity; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; InsP3, inositol 1,4,5-triphosphate; PericamR, ratiometric pericam; GFP, green fluorescent protein; KRB, Krebs-Ringer buffer; CaM, calmodulin. In recent years, these global changes have been dissected into regional variations, and different organelles have seen their importance accrued. In particular, in practically all cell types investigated, it has been found that the speed and amplitude of mitochondrial Ca2+ uptake depend not only on the amplitude of the [Ca2+]c rise, but also on the source of Ca2+ and the mechanisms through which the Ca2+ increase is elicited. Specifically, fast increase of mitochondrial Ca2+ concentration ([Ca2+]m) can be triggered by either Ca2+ mobilization from stores or Ca2+ influx from the medium, or both, depending on the cell type. Because mitochondrial Ca2+ uptake takes place exclusively through the so-called calcium uniporter, and because this uniporter has a low affinity for Ca2+, it was not clear how the relatively low, average [Ca2+]c increase elicited by physiological stimuli could efficiently drive the observed rise in [Ca2+]m. The concept of high Ca2+ microdomains was thus developed to explain the very rapid Ca2+ uptake by mitochondria under physiological conditions. The hypothesis predicts that the fast [Ca2+]m increases depend on the close vicinity of these organelles to Ca2+ channels, where the Ca2+ concentration is sufficiently high to drive an efficient uptake by the low affinity mitochondrial uniporter (1Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (985) Google Scholar, 2Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1748) Google Scholar). It is worth noting that this microdomain model does not purport to explain all mitochondrial Ca2+ uptake; rather, the model provides a conceptual framework to explain the fast [Ca2+]m rises, erst-while paradoxical. Given the motility of both ER and mitochondria, and the requirement for regions of close proximity between the two organelles to ensure a highly efficient mitochondrial Ca2+ uptake, it becomes of paramount importance to understand how this spatial arrangement is obtained. From a structural point of view, the ER is a highly convoluted reticular network, while mitochondria have historically been erroneously depicted as "fuse-like" individual organelles that pepper the cytoplasm. The notion that also mitochondria form a network within the cytoplasm is practically half a century old, but only recently has it been more widely accredited, with in vivo studies that have revealed a highly dynamic network that continuously undergoes multiple fusion and fission processes (2Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1748) Google Scholar, 3Johnson L.V. Walsh M.L. Chen L.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 990-994Crossref PubMed Scopus (1267) Google Scholar, 4Bereiter-Hahn J. Int. Rev. Cytol. 1990; 122: 1-63Crossref PubMed Scopus (275) Google Scholar, 5Bereiter-Hahn J. Vöth M. Microsc. Res. Tech. 1994; 27: 198-219Crossref PubMed Scopus (709) Google Scholar). Essentially, two major scenarios can be envisaged. On one hand, the vicinity of mitochondria to Ca2+ release sites may be a stochastic event, because of the abundance and motility of both organelles. On the other, transient or permanent interactions may exist to keep specific mitochondrial subpopulations close to sites where Ca2+ reaches high concentrations. It has been suggested that stable mitochondria-ER interactions might occur in adrenal medullary cells (6Montero M. Alonso M.T. Carnicero E. Cuchillo-Ibáñez I. Albillos A. Garcia A.G. García-Sancho J. Alvarez J. Nat. Cell Biol. 2000; 2: 57-61Crossref PubMed Scopus (411) Google Scholar), but this phenomenon has not been analyzed in detail. In the present study, we adopted a bipartite approach to explore this issue. For single cell studies we employed the new GFP-based Ca2+ probes selectively targeted to the mitochondrial matrix, whereas at the cell population level we took advantage of specific characteristics of aequorin, a Ca2+ probe with an established track record. Generation of Constructs—cDNA encoding the N-terminal part (comprising the first 36 amino acids) of subunit VIII of human cytochrome c oxidase was fused, in-frame, to cDNA encoding DsRed (Clontech) to generate mt-DsRed. Mitochondrially targeted versions of Cameleon and split Cameleons were generated similarly. Cameleon, split Cameleons, and mtCamgaroo-2, and mt- and nuPericamR were generous gifts from Roger Y. Tsien and Atsushi Miyawaki, respectively. Details of all constructs are available upon request. Cell Cultures and Transfection—HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, supplemented with l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml), in a humidified atmosphere containing 5% CO2. For transient transfections, cells were seeded onto glass coverslips (24-and 13-mm diameter for single cell imaging and for aequorin measurements, respectively). Transfections were performed at 50-70% confluence with the calcium phosphate method, using a total of 4 or 10 μg of DNA for small or large coverslips, respectively. Cell Imaging—Cells expressing fluorescent probes were observed 36 h after transfection on an inverted fluorescence microscope (Zeiss Axioplan), with an oil immersion objective (×63, N.A. 1.40). Excitation light at appropriate wavelengths was produced by a monochromator (Polychrome II, TILL Photonics, Martinsried, Germany): 440 nm for cameleon and CFP-CaM, 500 nm for M13-YFP and camgaroo, and 415 and 490 nm for PericamR. Dichroic beam splitters were 455DRLP, 525DRLP, and 505DRLP, respectively. Emission filters were 480DF30 (for CFP) and 545DF35 (for YFP) in the case of cameleon, HQ520LP in the case of camgaroo, and 535RDF45 in the case of PericamR; when using cameleon, the emission filters were alternated using a filter wheel (Lambda 10-2, Sutter Instruments, San Rafael, CA). Filters and dichroic beam splitters were purchased from Omega Optical and Chroma Technologies (Brattleboro, VT). Images were acquired using a cooled CCD camera (Imago, TILL Photonics) attached to a 12-bit frame grabber. Synchronization of the monochromator and CCD camera was performed through a control unit using TILLvisION version 4.0 (TILL Photonics); this software was also used for image analysis. Additional image analyses employed the public domain ImageJ program (developed at the United States National Institutes of Health by Wayne Rasband and available on the Internet. 2http://rsb.info.nih.gov/ij/. For co-localization studies, confocal planes were obtained using a Bio-Rad MRC1024ES system: for mtPericamR, the 488-nm line of a Kr-Ar laser was used together with a 522DF35 emission filter, whereas for mtDsRed, the 568-nm line and a 605DF32 emission filter were used. Unless otherwise specified, F measurements refer to average pixel intensities in regions covering >50% of the total mitochondrial or nuclear area. Whereas camgaroos respond to Ca2+ changes only with a change in F, ratiometric pericam shows an antiparallel behavior in response to Ca2+, i.e. upon Ca2+ binding, F upon excitation at 415 nm decreases, whereas F upon excitation at 490 nm increases. In a few experiments, however, the 415- and 490-nm signals did not behave in a perfectly antiparallel way. Such a behavior was random and most often because of either photobleaching or movement. In these cases the ratio of the two signals nicely corrects for the artifact. In a few cases, however, we noticed a drop of the 490-nm signal not accompanied by an increase in the 415-nm fluorescence that were not attributable to the above artifacts. The reason for this rare behavior is presently unknown, but, if it is observed, it can be verified by monitoring the fluorescence at the isosbestic point (470 nm). Aequorin Measurements—Cells transiently expressing aequorin were reconstituted in Krebs-Ringer buffer (KRB; containing, in mm, 125 NaCl, 5 KCl, 1 Na3PO4, 1 MgSO4, 5.5 glucose, 20 Hepes, pH 7.4, at 37 °C), supplemented with 1 mm CaCl2 and 5 μm coelenterazine (Molecular Probes, Leiden, The Netherlands) for 2 h. During the experimental procedure, cells were placed in a temperature-controlled chamber and perfused with KRB. Photons emitted were collected and analyzed as previously described (22Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Cell Stimulations—For in situ calibration experiments using mtPericamR, permeabilization was carried out by incubating cells in modified KRB (mKRB; containing, in mm, 10 NaCl, 130 KCl, 1 Na3PO4, 1 MgS04, 5 succinate, 10 Tris, pH 8.0, at 37 °C), supplemented with digitonin (100 μm) and FCCP (4 μm), after permeabilization. For in situ calibration experiments using nuPericamR, mKRB was buffered not with Tris but with 20 mm Hepes, pH 7.0, at 37 °C, and supplemented only with digitonin (20 μm); in addition, Staphylococcus aureus α-toxin was used (Sigma; 100 μg/ml); cells were maintained in mKRB for the remaining experimental procedures. Perfusion with histamine (100 μm) was employed to trigger Ca2+ release from intracellular stores. Perfusion with cyclopiazonic acid (20 μm) was employed to inhibit ER Ca2+-ATPase. For cytoskeleton disruption, cells were first challenged with histamine and then incubated in KRB, in the presence of colchicine (ICN, Milan, Italy; 10 μg/ml) or cytochalasin D (ICN; 10 μm), or both, for 30 min. The second histamine stimulus was applied in the presence of the same drug(s). All experimental procedures and incubations were carried out at 37 °C. All chemicals were of analytical or highest available grade and, unless otherwise stated, were acquired from Sigma. Data shown represent typical results obtained in at least five independent experiments; numerical data are presented as mean ± S.D.; statistical significance was calculated by applying Student's t test. Choice of a Mitochondrial Ca 2+ Indicator for Single Cell Studies—The positively charged fluorescent indicator rhod-2 is often considered the indicator of choice to measure changes in mitochondrial [Ca2+] at the single cell level. Although this probe has been extensively used by different groups, its subcellular localization is far from ideal; most likely because of this, its use has given rise to partially contradictory results in different cell types (7Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Hajnóczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (943) Google Scholar, 9Trollinger D.R. Cascio W.E. Lemasters J.J. Biophys. J. 2000; 79: 39-50Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Kaftan E.J. Xu T. Abercrombie R.F. Hille B. J. Biol. Chem. 2000; 275: 25465-25470Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The search for alternative calcium indicators has prompted the development of a number of genetically encoded fluorescent probes. These probes, however, have yet to be thoroughly characterized. Below we consider the three families of fluorescent indicators that have been recently introduced, i.e. Cameleons, Camgaroos, and Pericams. Cameleon is a GFP-based Ca2+ indicator, which was initially introduced as a probe for Ca2+ in the cytoplasm, ER, and nucleus (11Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2573) Google Scholar). A mitochondrial cameleon has been generated by fusing at the N terminus of the cytosolic construct the targeting sequence of subunit VIII of human cytochrome c oxidase (12Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). This particular mitochondrial targeting presequence has been repeatedly shown to deliver fused proteins in an highly efficient and selective manner to the mitochondrial matrix (13Rizzuto R. Simpson A.W.M. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (774) Google Scholar, 14Brini M. Pinton P. King M.P. Davidson M. Schon E.A. Rizzuto R. Nat. Med. 1999; 5: 951-954Crossref PubMed Scopus (138) Google Scholar). In a series of elegant experiments (12Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), it was observed that a cameleon targeted to mitochondria by this strategy only localizes efficiently in cells that express very low levels of the construct. We also confirmed that this fusion protein fails to localize correctly and transfected cells exhibit fluorescence throughout the cytoplasm; only rarely was a selective labeling of mitochondria observed. An attempt to bypass this problem was to use the so-called split cameleon (11Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2573) Google Scholar), in which two functional constituents of the cameleon are expressed individually. The two halves of the indicator were targeted to the mitochondrial matrix independently, using the same presequence as before. In this case, the M13-YFP moiety was efficiently delivered exclusively to the mitochondrial matrix (Fig. 1A, left panel); targeting of the CFP-CaM part was again deficient, although, with ∼50% of the fluorescence present throughout the cell body (Fig. 1A, right panel). The reasons for this behavior are not clear at present. Because of these difficulties, no further attempt to characterize mtCameleons was undertaken. Camgaroo is an insertional mutant of GFP, sensitive to Ca2+ (15Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11241-11246Crossref PubMed Scopus (717) Google Scholar). A mitochondrial version of this probe (mtCamgaroo-2; Ref. 16Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar) possesses an N-terminal presequence that effectively targets it to the mitochondrial matrix (Fig. 1B), although in strongly fluorescent cells a residual signal is often present throughout the cell cytoplasm. Results obtained by Tsien and co-workers (16Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar) indicated that mtCamgaroo-2 can be used to monitor changes in [Ca2+]m. Surprisingly, in our hands, HeLa cells expressing mtCamgaroo-2 transiently showed marginal, often undetected, changes in fluorescence when challenged with histamine (see below). The reason for this discrepancy lies in the illumination protocol used. When cells were illuminated continuously, a very rapid drop (to ∼50% of the initial F) was observed; this drop was not due to photobleaching because a short recovery (30 s) in the dark resulted in almost complete recovery of the initial F (Fig. 2A, upper panel). Importantly, the photoconverted form of the probe was almost completely insensitive to Ca2+ changes, as indicated by the lack of increase in F upon histamine addition after ∼10 s of continuous illumination (Fig. 2A, upper panel). If, on the contrary, the time of illumination was reduced to a minimum and at least 1 s between two successive illuminations were allowed, a clear increase in F was revealed by mtCamgaroo-2 upon histamine challenge (Fig. 2A, lower panel), on average about 20% of the initial value. Under the same conditions, a non-Ca2+-sensitive, but pH-sensitive, YFP gave no change in signal (Fig. 2A, lower panel). Using mtCamgaroo-2, we observed a substantial subcellular heterogeneity in peak Ca2+ increases in single HeLa cells challenged with histamine (Fig. 2B), in agreement with previous studies with rhod-2 or mtCameleons (7Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Arnaudeau S. Kelley W.L. Walsh Jr., J.V. Demaurex N. J. Biol. Chem. 2001; 276: 29430-29439Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). With probes such as rhod-2 or mtCamgaroo-2 (that respond to alterations in [Ca2+] simply by changing F), changes in size and shape of organelles, as well as organelle movement, may all create artifacts that confound both quantitative and qualitative analyses (17Dunn K.W. Mayor S. Myers J.N. Maxfield F.R. FASEB J. 1994; 8: 573-582Crossref PubMed Scopus (98) Google Scholar). To overcome these limitations, we shifted our attention to the most recent genetically targeted Ca2+ probe family (the Pericams; Ref. 18Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar). Fig. 1C shows the typical staining pattern of a HeLa cell transiently expressing a mitochondrially targeted ratiometric pericam (mtPericamR). The staining is highly specific and completely overlaps with that of a mitochondrially targeted red variant of GFP (mtDsRed). In addition, the nuF is almost indistinguishable from the background of non-transfected cells. The use of mtPericamR greatly diminishes the problem of photoconversion that plagues mtCamgaroo-2. Upon almost continuous illumination there is still an initial, rapid decrease in F at both wavelengths (but only ∼15%); as in the case of mtCamgaroo-2, the initial F is recovered after brief periods of non-illumination (data not shown). The significant difference in relation to mtCamgaroo-2 is that this probe retains its Ca2+ sensitivity even during periods of rapid data acquisition (see below). Characterization of mtPericamR—HeLa cells expressing mt-PericamR and challenged with histamine show a subcellular heterogeneous response, as reported previously using other non-ratiometric indicators, confirming that the differences between mitochondrial populations are real and not artifacts of movement. Fig. 3A shows pseudocolor-rendered ratios of a HeLa cell before and after a histamine challenge. When different subsets of mitochondria are selected for analyses, a clear heterogeneity is observed (Fig. 3B). We next addressed the problem of the relationship between mitochondrial and cytoplasmic Ca2+ changes in the same cell. Given that it has been repeatedly demonstrated in HeLa cells that the nucleus behaves essentially as the cytoplasm, we cotransfected these cells with a variant of PericamR targeted to the nucleus (nuPericamR) and mtPericamR. Under resting conditions, the ratio value was higher in the latter. This most likely reflects a difference in resting pH, rather than in [Ca2+] (see below). Upon a histamine challenge both compartments showed similar kinetics: a sharp rise in the F ratio, followed by a declining plateau, often accompanied by oscillations (Fig. 3C). Quantitatively, it was surprising to note that this increase was larger in the nucleus than in the mitochondria. This result is in sharp contrast with previous data obtained with aequorin, rhod-2, and mtCameleon, with all these probes, the increase in [Ca2+] has been found to be larger within mitochondria than in the nucleus (and cytoplasm). To verify whether this contradiction was because of a different affinity of the probe for Ca2+ in the two compartments, we carried out a calibration in situ with the widely used ionomycin and high Ca2+ method (e.g. Ref. 16Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar). As shown in Fig. 4A (upper panel), both compartments exhibit a sharp increase in the ratio upon histamine stimulation, which returns to basal levels after the agonist is washed away; addition of Ca2+ in the presence of the ionophore also causes an increase in the ratio, this time clearly biphasic: a first rapid increase, followed by a larger and slower rise. However, analysis of the two individual wavelengths shows distinct pictures in the case of histamine and ionomycin. In the former, the increase in ratio is because of a small increase in F upon excitation at 490 nm and a larger decrease in F upon excitation at 415 nm (Fig. 4A, lower panel). In the latter, the first rise is similar to that observed for histamine; the second rise, however, is fully because of an increase in F upon excitation at 490 nm, whereas the 415-nm signal remains essentially unchanged. Given that alkalinization increases the F of PericamR at 490 nm (18Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar), the possibility of a pH artifact in the effect of ionomycin was thus considered. Cells expressing nuPericamR and mtPericamR were challenged with NH4Cl to increase the pH inside the cells without grossly changing the [Ca2+] (verified with fura-2). An apparent increase in [Ca2+] was observed in both compartments (Fig. 4B, upper panel), but this was due simply to an increase in the 490-nm signal (Fig. 4B, lower panel). A further confirmation that the ionomycin effect is because of a pH artifact is provided by monitoring F at the isosbestic point (470 nm). As expected, no fluorescence increase was observed at this wavelength in the case of histamine, whereas both ionomycin and NH4Cl caused a major increase in F at this wavelength (data not shown). To obtain an in situ measurement of the Ca2+ affinity of mtPericamR, we employed the following protocol. Cells were first treated with digitonin to permeabilize the plasma membrane and then with the mitochondrial uncoupler FCCP to equilibrate matrix pH with that in the medium. The external medium, without added Ca2+ and supplemented with 100 μm EGTA, was buffered at pH 8 to mimic the conditions of the mitochondrial matrix in intact cells. Under these conditions, given the absence of a mitochondrial membrane potential and of a pH gradient, matrix [Ca2+] rapidly establishes an equilibrium with the extracellular [Ca2+] via the uniporter. Indeed, further addition of ionomycin at any [Ca2+] resulted in no appreciable change in F. Increasing concentrations of CaCl2 in the medium resulted in increased F ratios (Fig. 5A) with no change at the isosbestic point. The apparent K′d of mtPericamR for Ca2+ was thus determined to be ∼11 μM (Fig. 5B), much higher than the 1.3 μm calculated in vitro (18Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar). Using this calibration procedure we could conclude that the mean increase in mitochondrial [Ca2+] at the peak of a histamine challenge is about 10 μm with some regions reaching values well over 50 μm (see, for example, Fig. 3A, where ratio values of 2.5 and 3.4 correspond to 10 and 50 μm, respectively, according to the calibration shown in Fig. 5, A and B). It was more difficult to perform the same calibration procedure in the nucleus, because the digitonin treatment (100 μm, as used for calibrating mtPericamR) generally provoked the release of the nuclear probe. We tried to permeabilize the cells with S. aureus α-toxin (19Bhakdi S. Weller U. Walev I. Martin E. Jonas D. Palmer M. Med. Microbiol. Immunol. 1993; 182: 167-175Crossref PubMed Scopus (205) Google Scholar) but the results were unsatisfactory, given that very high concentrations of the toxin (∼100 μg/ml) resulted only in partial release of a trapped fluorescence probe such as fura-2. However, at lower digitonin concentrations (20 μm), complete release of fura-2 was obtained, whereas in the majority of the cells a large part of nuPericamR remained localized in the nucleus. Accordingly, the K′d of nuPericamR in situ was calculated to be ∼2.5 μm, similar to that calculated in vitro. The kinetics of the mitochondrial Ca2+ increase with respect to that in the nucleus were analyzed in the experiments presented in Fig. 6. The high rate of data acquisition permitted by the use of PericamR enabled us to acquire data points (F ratio upon excitation at two independent wavelengths) every 180-200 ms. Detailed analyses of the ratio increases immediately following histamine stimulation revealed different types of heterogeneity. In some cells, the mitochondrial response time was homogeneous, but with different rates of Ca2+ accumulation (Fig. 6A). In others, mitochondria have similar rates of Ca2+ uptake, but different delays in relation to the nuclear [Ca2+] rise (data not shown). In general, the heterogeneity observed among different mitochondrial populations, in terms of the lag time between nucleoplasmic and mitochondrial Ca2+ increases, ranged from not appreciable (less than 200 ms) to well above 400 ms. On the other hand, in accordance with previously published results (18Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar), the peak response of the mitochondria was reached with some delay (3-5 s) with respect to that in the nucleus (1-2 s). Stochastic or Regulated ER-Mitochondria Interactions?—Experiments presented above and previously published data confirm a substantial heterogeneity in the mitochondrial response to the Ca2+ increases elicited by an agent such as histamine (that by producing InsP3, mobilizes Ca2+ from intracellular stores). The accepted interpretation for such heterogeneity (and for the speed and amplitude of mitochondrial Ca2+ increases) is that some of the organelles are very close to the InsP3-gated channels. In this way, they are exposed not to the mean increases in cytoplasmic [Ca2+], but rather to the microdomains of much higher [Ca2+] that are formed close to the channels themselves. The still unanswered, but key question, is whether this vicinity is simply a stochastic event of two organelles that are densely packed within the cell (whereby the random movement and reorganization of
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