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Mitochondria Recycle Ca2+ to the Endoplasmic Reticulum and Prevent the Depletion of Neighboring Endoplasmic Reticulum Regions*210

Open AccessPublished:August 01, 2001DOI:https://doi.org/10.1074/jbc.M103274200
      To study Ca2+ 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 ± 5 µm, indicating that Ca2+ transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca2+]ER averaged 371 ± 21 µm at rest and decreased to 133 ± 14 µm and 59 ± 5 µm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca2+ uptake was prevented by oligomycin and rotenone or when Ca2+ efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca2+ taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca2+ than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca2+ uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca2+ release and recycle a substantial portion of the captured Ca2+ back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca2+ signals and the filling state of neighboring ER regions.
      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
      The calcium ion is a ubiquitous intracellular messenger that controls processes ranging from fertilization and cellular differentiation to muscle contraction and synaptic transmission (
      • Clapham D.E.
      ,
      • Berridge M.J.
      • Bootman M.D.
      • Lipp P.
      ). 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 (
      • Nelson M.T.
      • Cheng H.
      • Rubart M.
      • Santana L.F.
      • Bonev A.D.
      • Knot H.J.
      • Lederer W.J.
      ,
      • Bito H.
      • Deisseroth K.
      • Tsien R.W.
      ,
      • Berridge M.
      • Lipp P.
      • Bootman M.
      ). Whereas the influx of calcium through voltage-dependent membrane channels triggers rapid secretion at synapses (), 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 (
      • Berridge M.J.
      ). These calcium oscillations can be decoded in the cytosol by frequency-sensitive effector proteins such as calmodulin-dependent kinase II (
      • De Koninck P.
      • Schulman H.
      ), and have been shown to optimize both secretion (
      • Tse A.
      • Tse F.W.
      • Almers W.
      • Hille B.
      ) and gene expression (
      • Dolmetsch R.E.
      • Xu K.
      • Lewis R.S.
      ,
      • Li W.
      • Llopis J.
      • Whitney M.
      • Zlokarnik G.
      • Tsien R.Y.
      ). Calcium oscillations can also be decoded by mitochondria (
      • Hajnoczky G.
      • Robb-Gaspers L.D.
      • Seitz M.B.
      • Thomas A.P.
      ), 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 (
      • Hajnoczky G.
      • Robb-Gaspers L.D.
      • Seitz M.B.
      • Thomas A.P.
      ,
      • Rizzuto R.
      • Bastianutto C.
      • Brini M.
      • Murgia M.
      • Pozzan T.
      ).
      In addition to being able to decode Ca2+ oscillations, mitochondria also participate actively in calcium signaling (reviewed in Refs.
      • Babcock D.F.
      • Hille B.
      ,
      • Duchen M.R.
      ,
      • Rutter G.A.
      • Rizzuto R.
      ). 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 (
      • Tinel H.
      • Cancela J.M.
      • Mogami H.
      • Gerasimenko J.V.
      • Gerasimenko O.V.
      • Tepikin A.V.
      • Petersen O.H.
      ). Importantly, mitochondria are often in close contact with Ca2+ release sites in the endoplasmic reticulum (
      • Simpson P.B.
      • Mehotra S.
      • Lange G.D.
      • Russell J.T.
      ,
      • Rizzuto R.
      • Pinton P.
      • Carrington W.
      • Fay F.S.
      • Fogarty K.E.
      • Lifshitz L.M.
      • Tuft R.A.
      • Pozzan T.
      ) or with Ca2+ influx channels at the plasma membrane (
      • Hoth M.
      • Fanger C.M.
      • Lewis R.S.
      ). By acting as a Ca2+ buffering system at these strategic locations, mitochondria can modulate the rate of Ca2+ release by IP3 receptors (
      • Babcock D.F.
      • Herrington J.
      • Goodwin P.C.
      • Park Y.B.
      • Hille B.
      ,
      • Hajnoczky G.
      • Hager R.
      • Thomas A.P.
      ) or the rate of capacitative Ca2+ entry through CRAC channels (
      • Hoth M.
      • Fanger C.M.
      • Lewis R.S.
      ). 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 (
      • Jouaville L.S.
      • Ichas F.
      • Holmuhamedov E.L.
      • Camacho P.
      • Lechleiter J.D.
      ,
      • Simpson P.B.
      • Russell J.T.
      ,
      • Boitier E.
      • Rea R.
      • Duchen M.R.
      ,
      • Kaftan E.J.
      • Xu T.
      • Abercrombie R.F.
      • Hille B.
      ).
      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 Mag-indo-1 have been used to measure calcium within the ER and mitochondria (
      • Hofer A.M.
      • Machen T.E.
      ,
      • Hirose K.
      • Iino M.
      ,
      • Tse A.
      • Tse F.W.
      • Hille B.
      ,
      • Golovina V.A.
      • Blaustein M.P.
      ,
      • Hofer A.M.
      • Fasolato C.
      • Pozzan T.
      ). 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 (
      • Hajnoczky G.
      • Robb-Gaspers L.D.
      • Seitz M.B.
      • Thomas A.P.
      ,
      • Hoth M.
      • Fanger C.M.
      • Lewis R.S.
      ,
      • Babcock D.F.
      • Herrington J.
      • Goodwin P.C.
      • Park Y.B.
      • Hille B.
      ,
      • Simpson P.B.
      • Russell J.T.
      ) 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 (
      • Rizzuto R.
      • Brini M.
      • Pozzan T.
      ,
      • Rizzuto R.
      • Brini M.
      • Pozzan T.
      ) and has been used extensively to measure calcium dynamics within the mitochondria (
      • Rizzuto R.
      • Bastianutto C.
      • Brini M.
      • Murgia M.
      • Pozzan T.
      ,
      • Rizzuto R.
      • Simpson A.W.
      • Brini M.
      • Pozzan T.
      ,
      • Rutter G.A.
      • Burnett P.
      • Rizzuto R.
      • Brini M.
      • Murgia M.
      • Pozzan T.
      • Tavare J.M.
      • Denton R.M.
      ,
      • Montero M.
      • Alonso M.T.
      • Carnicero E.
      • Cuchillo-Ibanez I.
      • Albillos A.
      • Garcia A.G.
      • Garcia-Sancho J.
      • Alvarez J.
      ), the ER (
      • Montero M.
      • Brini M.
      • Marsault R.
      • Alvarez J.
      • Sitia R.
      • Pozzan T.
      • Rizzuto R.
      ,
      • Button D.
      • Eidsath A.
      ,
      • Barrero M.J.
      • Montero M.
      • Alvarez J.
      ,
      • Alonso M.T.
      • Barrero M.J.
      • Michelena P.
      • Carnicero E.
      • Cuchillo I.
      • Garcia A.G.
      • Garcia-Sancho J.
      • Montero M.
      • Alvarez J.
      ,
      • Pinton P.
      • Ferrari D.
      • Magalhaes P.
      • Schulze-Osthoff K.
      • Di Virgilio F.
      • Pozzan T.
      • Rizzuto R.
      ), and the Golgi complex (
      • Pinton P.
      • Pozzan T.
      • Rizzuto R.
      ). 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 (
      • Miyawaki A.
      • Llopis J.
      • Heim R.
      • McCaffery J.M.
      • Adams J.A.
      • Ikura M.
      • Tsien R.Y.
      ,
      • Miyawaki A.
      • Griesbeck O.
      • Heim R.
      • Tsien R.Y.
      ) appear better suited for calcium measurements in organelles. The bright fluorescence of the green fluorescent protein mutants, combined with selective targeting sequences, allows one to visualize the calcium signals in organelles by fluorescence ratio imaging (
      • Fan G.Y.
      • Fujisaki H.
      • Miyawaki A.
      • Tsay R.K.
      • Tsien R.Y.
      • Ellisman M.H.
      ,
      • Emmanouilidou E.
      • Teschemacher A.G.
      • Pouli A.E.
      • Nicholls L.I.
      • Seward E.P.
      • Rutter G.A.
      ,
      • Yu R.
      • Hinkle P.M.
      ). Furthermore, the calcium affinity of calmodulin can be adjusted by molecular engineering, enabling one to match the calcium concentration within the organelle of interest (
      • Miyawaki A.
      • Llopis J.
      • Heim R.
      • McCaffery J.M.
      • Adams J.A.
      • Ikura M.
      • Tsien R.Y.
      ). 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 µm 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.

      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 µm in HeLa cells, values that agree well with previous reports using aequorin or cameleons (
      • Barrero M.J.
      • Montero M.
      • Alvarez J.
      ,
      • Pinton P.
      • Ferrari D.
      • Magalhaes P.
      • Schulze-Osthoff K.
      • Di Virgilio F.
      • Pozzan T.
      • Rizzuto R.
      ,
      • Miyawaki A.
      • Llopis J.
      • Heim R.
      • McCaffery J.M.
      • Adams J.A.
      • Ikura M.
      • Tsien R.Y.
      ,
      • Yu R.
      • Hinkle P.M.
      ,
      • Montero M.
      • Barrero M.J.
      • Alvarez J.
      ). Substantial ER refilling was observed during agonist stimulation even in the absence of extracellular Ca2+, consistent with a recent study using targeted cameleon (
      • Yu R.
      • Hinkle P.M.
      ). 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.6 b). 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 (
      • Rizzuto R.
      • Pinton P.
      • Carrington W.
      • Fay F.S.
      • Fogarty K.E.
      • Lifshitz L.M.
      • Tuft R.A.
      • Pozzan T.
      ), and that Ca2+ signal transmission between these organelles is quasisynaptic (
      • Csordas G.
      • Thomas A.P.
      • Hajnoczky G.
      ). Consistent with these observations, we observed that [Ca2+]mittransients reached submillimolar values in a fraction of mitochondria (Fig. 3), which must be very close to the sites of Ca2+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 low-affinity YC4.1mit probe, which reported an average peak [Ca2+]mit around 100 µm (Fig.3). This finding is consistent with recent imaging data obtained in HeLa cells with a permutated green fluorescent protein engineered to sense Ca2+ (
      • Nagai T.
      • Sawano A.
      • Park E.S.
      • Miyawaki A.
      ), 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 (
      • Montero M.
      • Alonso M.T.
      • Carnicero E.
      • Cuchillo-Ibanez I.
      • Albillos A.
      • Garcia A.G.
      • Garcia-Sancho J.
      • Alvarez J.
      ), possibly reflecting the close proximity of these mitochondria to both the plasma membrane and Ca2+ release channels (
      • Montero M.
      • Alonso M.T.
      • Carnicero E.
      • Cuchillo-Ibanez I.
      • Albillos A.
      • Garcia A.G.
      • Garcia-Sancho J.
      • Alvarez J.
      ). 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 (
      • Jouaville L.S.
      • Pinton P.
      • Bastianutto C.
      • Rutter G.A.
      • Rizzuto R.
      ). 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 (
      • Landolfi B.
      • Curci S.
      • Debellis L.
      • Pozzan T.
      • Hofer A.M.
      ,
      • Montero M.
      • Barrero M.J.
      • Alvarez J.
      ). 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 (
      • Gurney A.M.
      • Drummond R.M.
      • Fay F.S.
      ). 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+]mitchanges. 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 (
      • Gurney A.M.
      • Drummond R.M.
      • Fay F.S.
      ). The decreased [Ca2+] release observed with Mag-fura in the presence of mitochondrial inhibitors thus likely reflects the decreased [Ca2+]mitsignal, rather than the true [Ca2+]ERresponse (
      • Landolfi B.
      • Curci S.
      • Debellis L.
      • Pozzan T.
      • Hofer A.M.
      ). 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. 6 b).
      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 inhibited (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 (
      • Park M.K.
      • Petersen O.H.
      • Tepikin A.V.
      ). 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. Mühlethaler, and W. Graier for critical reading of the manuscript.

      REFERENCES

        • Clapham D.E.
        Cell. 1995; 80: 259-268
        • Berridge M.J.
        • Bootman M.D.
        • Lipp P.
        Nature. 1998; 395: 645-648
        • Nelson M.T.
        • Cheng H.
        • Rubart M.
        • Santana L.F.
        • Bonev A.D.
        • Knot H.J.
        • Lederer W.J.
        Science. 1995; 270: 633-637
        • Bito H.
        • Deisseroth K.
        • Tsien R.W.
        Cell. 1996; 87: 1203-1214
        • Berridge M.
        • Lipp P.
        • Bootman M.
        Curr. Biol. 1999; 9: R157-159
        • Neher E.
        Neuron. 1998; 20: 389-399
        • Berridge M.J.
        Nature. 1993; 361: 315-325
        • De Koninck P.
        • Schulman H.
        Science. 1998; 279: 227-230
        • Tse A.
        • Tse F.W.
        • Almers W.
        • Hille B.
        Science. 1993; 260: 82-84
        • Dolmetsch R.E.
        • Xu K.
        • Lewis R.S.
        Nature. 1998; 392: 933-936
        • Li W.
        • Llopis J.
        • Whitney M.
        • Zlokarnik G.
        • Tsien R.Y.
        Nature. 1998; 392: 936-941
        • Hajnoczky G.
        • Robb-Gaspers L.D.
        • Seitz M.B.
        • Thomas A.P.
        Cell. 1995; 82: 415-424
        • Rizzuto R.
        • Bastianutto C.
        • Brini M.
        • Murgia M.
        • Pozzan T.
        J. Cell Biol. 1994; 126: 1183-1194
        • Babcock D.F.
        • Hille B.
        Curr. Opin. Neurobiol. 1998; 8: 398-404
        • Duchen M.R.
        J. Physiol. 1999; 516: 1-17
        • Rutter G.A.
        • Rizzuto R.
        Trends Biochem. Sci. 2000; 25: 215-221
        • Tinel H.
        • Cancela J.M.
        • Mogami H.
        • Gerasimenko J.V.
        • Gerasimenko O.V.
        • Tepikin A.V.
        • Petersen O.H.
        EMBO J. 1999; 18: 4999-5008
        • Simpson P.B.
        • Mehotra S.
        • Lange G.D.
        • Russell J.T.
        J. Biol. Chem. 1997; 272: 22654-22661
        • Rizzuto R.
        • Pinton P.
        • Carrington W.
        • Fay F.S.
        • Fogarty K.E.
        • Lifshitz L.M.
        • Tuft R.A.
        • Pozzan T.
        Science. 1998; 280: 1763-1766
        • Hoth M.
        • Fanger C.M.
        • Lewis R.S.
        J. Cell Biol. 1997; 137: 633-648
        • Babcock D.F.
        • Herrington J.
        • Goodwin P.C.
        • Park Y.B.
        • Hille B.
        J. Cell Biol. 1997; 136: 833-844
        • Hajnoczky G.
        • Hager R.
        • Thomas A.P.
        J. Biol. Chem. 1999; 274: 14157-14162
        • Jouaville L.S.
        • Ichas F.
        • Holmuhamedov E.L.
        • Camacho P.
        • Lechleiter J.D.
        Nature. 1995; 377: 438-441
        • Simpson P.B.
        • Russell J.T.
        J. Biol. Chem. 1996; 271: 33493-33501
        • Boitier E.
        • Rea R.
        • Duchen M.R.
        J. Cell Biol. 1999; 145: 795-808
        • Kaftan E.J.
        • Xu T.
        • Abercrombie R.F.
        • Hille B.
        J. Biol. Chem. 2000; 275: 25465-25470
        • Hofer A.M.
        • Machen T.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2598-2602
        • Hirose K.
        • Iino M.
        Nature. 1994; 372: 791-794
        • Tse A.
        • Tse F.W.
        • Hille B.
        J. Physiol. 1994; 477: 511-525
        • Golovina V.A.
        • Blaustein M.P.
        Science. 1997; 275: 1643-1648
        • Hofer A.M.
        • Fasolato C.
        • Pozzan T.
        J. Cell Biol. 1998; 140: 325-334
        • Rizzuto R.
        • Brini M.
        • Pozzan T.
        Cytotechnology. 1993; 11: S44-46
        • Rizzuto R.
        • Brini M.
        • Pozzan T.
        Methods Cell Biol. 1994; 40: 339-358
        • Rizzuto R.
        • Simpson A.W.
        • Brini M.
        • Pozzan T.
        Nature. 1992; 358: 325-327
        • Rutter G.A.
        • Burnett P.
        • Rizzuto R.
        • Brini M.
        • Murgia M.
        • Pozzan T.
        • Tavare J.M.
        • Denton R.M.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494
        • Montero M.
        • Alonso M.T.
        • Carnicero E.
        • Cuchillo-Ibanez I.
        • Albillos A.
        • Garcia A.G.
        • Garcia-Sancho J.
        • Alvarez J.
        Nat. Cell Biol. 2000; 2: 57-61
        • Montero M.
        • Brini M.
        • Marsault R.
        • Alvarez J.
        • Sitia R.
        • Pozzan T.
        • Rizzuto R.
        EMBO J. 1995; 14: 5467-5475
        • Button D.
        • Eidsath A.
        Mol. Biol. Cell. 1996; 7: 419-434
        • Barrero M.J.
        • Montero M.
        • Alvarez J.
        J. Biol. Chem. 1997; 272: 27694-27699
        • Alonso M.T.
        • Barrero M.J.
        • Michelena P.
        • Carnicero E.
        • Cuchillo I.
        • Garcia A.G.
        • Garcia-Sancho J.
        • Montero M.
        • Alvarez J.
        J. Cell Biol. 1999; 144: 241-254
        • Pinton P.
        • Ferrari D.
        • Magalhaes P.
        • Schulze-Osthoff K.
        • Di Virgilio F.
        • Pozzan T.
        • Rizzuto R.
        J. Cell Biol. 2000; 148: 857-862
        • Pinton P.
        • Pozzan T.
        • Rizzuto R.
        EMBO J. 1998; 17: 5298-5308
        • Miyawaki A.
        • Llopis J.
        • Heim R.
        • McCaffery J.M.
        • Adams J.A.
        • Ikura M.
        • Tsien R.Y.
        Nature. 1997; 388: 882-887
        • Miyawaki A.
        • Griesbeck O.
        • Heim R.
        • Tsien R.Y.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2135-2140
        • Fan G.Y.
        • Fujisaki H.
        • Miyawaki A.
        • Tsay R.K.
        • Tsien R.Y.
        • Ellisman M.H.
        Biophys. J. 1999; 76: 2412-2420
        • Emmanouilidou E.
        • Teschemacher A.G.
        • Pouli A.E.
        • Nicholls L.I.
        • Seward E.P.
        • Rutter G.A.
        Curr. Biol. 1999; 9: 915-918
        • Yu R.
        • Hinkle P.M.
        J. Biol. Chem. 2000; 275: 23648-23653
        • Kim J.H.
        • Johannes L.
        • Goud B.
        • Antony C.
        • Lingwood C.A.
        • Daneman R.
        • Grinstein S.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2997-3002
        • Wu M.M.
        • Llopis J.
        • Adams S.
        • McCaffery J.M.
        • Kulomaa M.S.
        • Machen T.E.
        • Moore H.P.
        • Tsien R.Y.
        Chem. Biol. 2000; 7: 197-209
        • Landolfi B.
        • Curci S.
        • Debellis L.
        • Pozzan T.
        • Hofer A.M.
        J. Cell Biol. 1998; 142: 1235-1243
        • Mohr F.C.
        • Fewtrell C.
        Am. J. Physiol. 1990; 258: C217-226
        • Fulceri R.
        • Bellomo G.
        • Mirabelli F.
        • Gamberucci A.
        • Benedetti A.
        Cell Calcium. 1991; 12: 431-439
        • Montero M.
        • Barrero M.J.
        • Alvarez J.
        FASEB J. 1997; 11: 881-885
        • Csordas G.
        • Thomas A.P.
        • Hajnoczky G.
        EMBO J. 1999; 18: 96-108
        • Nagai T.
        • Sawano A.
        • Park E.S.
        • Miyawaki A.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202
        • Jouaville L.S.
        • Pinton P.
        • Bastianutto C.
        • Rutter G.A.
        • Rizzuto R.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13807-13812
        • Gurney A.M.
        • Drummond R.M.
        • Fay F.S.
        Cell Calcium. 2000; 27: 339-351
        • Park M.K.
        • Petersen O.H.
        • Tepikin A.V.
        EMBO J. 2000; 19: 5729-5739