Sustained Activity of Calcium Release-activated Calcium Channels Requires Translocation of Mitochondria to the Plasma Membrane

A rise of the intracellular Ca2+ concentration has multiple signaling functions. Sustained Ca2+ influx across plasma membrane through calcium release-activated calcium (CRAC) channels is required for T-cell development in the thymus, gene transcription, and proliferation and differentiation of naïve T-cells into armed effectors cells. Intracellular Ca2+ signals are shaped by mitochondria, which function as a highly dynamic Ca2+ buffer. However, the precise role of mitochondria for Ca2+-dependent T-cell activation is unknown. Here we have shown that mitochondria are translocated to the plasma membrane as a consequence of Ca2+ influx and that this directed movement is essential to sustain Ca2+ influx through CRAC channels. The decreased distance between mitochondria and the plasma membrane enabled mitochondria to take up large amounts of inflowing Ca2+ at the plasma membrane, thereby preventing Ca2+-dependent inactivation of CRAC channels and sustaining Ca2+ signals. Inhibition of kinesin-dependent mitochondrial movement along microtubules abolished mitochondrial translocation and reduced sustained Ca2+ signals. Our results show how a directed movement of mitochondria is used to control important cellular functions such as Ca2+-dependent T-cell activation. A rise of the intracellular Ca2+ concentration has multiple signaling functions. Sustained Ca2+ influx across plasma membrane through calcium release-activated calcium (CRAC) channels is required for T-cell development in the thymus, gene transcription, and proliferation and differentiation of naïve T-cells into armed effectors cells. Intracellular Ca2+ signals are shaped by mitochondria, which function as a highly dynamic Ca2+ buffer. However, the precise role of mitochondria for Ca2+-dependent T-cell activation is unknown. Here we have shown that mitochondria are translocated to the plasma membrane as a consequence of Ca2+ influx and that this directed movement is essential to sustain Ca2+ influx through CRAC channels. The decreased distance between mitochondria and the plasma membrane enabled mitochondria to take up large amounts of inflowing Ca2+ at the plasma membrane, thereby preventing Ca2+-dependent inactivation of CRAC channels and sustaining Ca2+ signals. Inhibition of kinesin-dependent mitochondrial movement along microtubules abolished mitochondrial translocation and reduced sustained Ca2+ signals. Our results show how a directed movement of mitochondria is used to control important cellular functions such as Ca2+-dependent T-cell activation. Ca2+ entry through calcium release-activated calcium (CRAC) 2The abbreviations used are: CRAC, Ca2+ release-activated Ca2+; BTP2, bis(trifluoromethyl)pyrazole 2; TG, thapsigargin; BAPTA, 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid; mAb, monoclonal antibody; NFAT, nuclear factor of activated T-cells. 2The abbreviations used are: CRAC, Ca2+ release-activated Ca2+; BTP2, bis(trifluoromethyl)pyrazole 2; TG, thapsigargin; BAPTA, 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid; mAb, monoclonal antibody; NFAT, nuclear factor of activated T-cells. channels, which are opened following depletion of Ca2+ stores, is necessary for T-cell activation (1Lewis R.S. Cahalan M.D. Annu. Rev. Immunol. 1995; 13: 623-653Crossref PubMed Scopus (447) Google Scholar, 2Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1788) Google Scholar, 3Quintana A. Griesemer D. Schwarz E.C. Hoth M. 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A. 2006; 103: 4040-4045Crossref PubMed Scopus (276) Google Scholar), and WAVE2, which is also involved in CRAC channel activation, probably through an interaction with the cytoskeleton (13Nolz J.C. Gomez T.S. Zhu P. Li S. Medeiros R.B. Shimizu Y. Burkhardt J.K. Freedman B.D. Billadeau D.D. Curr. Biol. 2006; 16: 24-34Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Apart from the major role of mitochondria as cellular energy sources and their role in cell death (14Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 15Duchen M.R. J. Physiol. (Lond.). 2000; 529: 57-68Crossref Scopus (915) Google Scholar), mitochondria have been identified as regulatory elements for CRAC channels (2Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1788) Google Scholar, 16Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (234) Google Scholar, 17Glitsch M.D. Bakowski D. Parekh A.B. EMBO J. 2002; 21: 6744-6754Crossref PubMed Scopus (177) Google Scholar). Mitochondria can reduce Ca2+-dependent inactivation of CRAC channels in submembranous Ca2+ microdomains by quickly removing the inflowing Ca2+, highlighting the importance of organelle localization within a cell. Mitochondrial localization and movements are determined by microtubules (18Ball E.H. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 123-126Crossref PubMed Scopus (159) Google Scholar, 19Knowles M.K. Guenza M.G. Capaldi R.A. Marcus A.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14772-14777Crossref PubMed Scopus (46) Google Scholar, 20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar, 21Varadi A. Johnson-Cadwell L.I. Cirulli V. Yoon Y. Allan V.J. Rutter G.A. J. Cell Sci. 2004; 117: 4389-4400Crossref PubMed Scopus (192) Google Scholar) and microfilaments (20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar, 22Morris R.L. Hollenbeck P.J. J. Cell Biol. 1995; 131: 1315-1326Crossref PubMed Scopus (440) Google Scholar). Recently, Ca2+ signaling and mitochondrial movements have been linked through an unknown Ca2+ sensor molecule that translates Ca2+ signals into the microtubular motor protein-based mitochondrial movements (20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar, 23Yoon Y. Science's STKE. 2005; 2005: pe18PubMed Google Scholar). These data suggest that mitochondrial movements may not always be random but could be directed to certain cellular domains and have specific localization-dependent functions. Cells—Human Jurkat T-cell lines were isolated and grown as described previously (24Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1995; 131: 655-667Crossref PubMed Scopus (164) Google Scholar, 25Schwarz A. Tutsch E. Ludwig B. Schwarz E.C. Stallmach A. Hoth M. J. Biol. Chem. 2004; 279: 5641-5647Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Reagents—All chemicals and antibodies not specifically mentioned were from Sigma (highest grade). Other reagents used in our experiments included Fura-2/AM, thapsigargin (TG), MitoTracker® Green FM, BAPTA (all from Molecular Probes), mouse anti-human CD3-RPE-conjugated mAb (DakoCytomation), mouse anti-human CD45-Alexa Fluor®488 mAb (Serotec), di-8-aminonaphthylethenylpyridinium (Invitrogen), and BTP2 (Altana Pharma). Fluorescence Microscopy and Ca2+ Imaging—Imaging experiments were done as previously described (26Philipp S. Strauss B. Hirnet D. Wissenbach U. Mery L. Flockerzi V. Hoth M. J. Biol. Chem. 2003; 278: 26629-26638Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). A 40× (Uplan/Apo, numerical aperture 1.0) or 100× (Uplan/Apo numerical aperture 1.35, oil immersion) objective were used. Ca2+ Ringer's solution contained (in mm): 155 NaCl, 4.5 KCl, 1 CaCl2, 2 MgCl2, 10 d-glucose, and 5 Hepes (pH 7.4 with NaOH). CaCl2 was substituted by 1 mm EGTA to prepare Ca2+-free Ringer's solution. To visualize mitochondria, cells were incubated with 100-200 nm MitoTracker® for 30 min at 22-23 °C. Excess dye was removed by washing twice. Cells were illuminated at 490 nm. DCLP 500 (UV) was used as a dichroic mirror and LP 515 as an emission filter. Bead Stimulation—As described previously (27Quintana A. Hoth M. Cell Calcium. 2004; 36: 99-109Crossref PubMed Scopus (20) Google Scholar), we followed the standard procedure for absorbing proteins on polystyrene microparticles (size >0.5 μm) established by Polysciences Europe GmbH Company (www.polysciences.com). Two alterations in the procedure were required to optimize our results. In step 1, we employed 100 μl of a 2.5% suspension of beads (∼ 2.1 × 107 beads), and in step 7, we added 50 μg of the protein to be absorbed. Chemical composition and pH of buffers were the same as recommended in the protocol. Azid-free anti-human CD3 mAbs (Euroclone) were passively coupled to microparticles (diameter = 5.83 μm). They were stored at 4 °C in the specified storage buffer until use. Beads were washed twice with phosphatebuffered saline before resuspending them in the Ringer's solution used for the experiments. Confocal Microscopy—For confocal imaging, a Nipkow discbased scanning head (QLC-100, VisiTech International) was attached to an upright microscope (Eclipse 600, Nikon) equipped with a 100× water lens (numerical aperture 1.1, Nikon). The light source was a 488-nm solid-state laser (Saphire 488-30, Coherent). A dichroic mirror between the microlens and pinhole disc reflects the emission light that passes a 500-nm long-pass barrier filter. For detection, a charge-coupled device camera (OrcaER, Hamamatsu Photonics) in the 2 × 2 binning mode, resulting in a × was used. was by the data at a of were of were same as previously described was used E. Griesemer D. Schwarz A. Stallmach A. Hoth M. J. Immunol. 2004; PubMed Scopus Google Scholar). The emission contained a dichroic mirror at nm followed by a channel 1 at nm and a channel 2 at 500 nm. The was nm for with nm. of the were 5 min with a of × or × To mitochondria, cells were with nm MitoTracker® for 1 The plasma membrane was with a mouse anti-human CD45-Alexa Fluor®488 Cells were incubated for 30 min with experiments were done as described previously (26Philipp S. Strauss B. Hirnet D. Wissenbach U. Mery L. Flockerzi V. Hoth M. J. Biol. Chem. 2003; 278: 26629-26638Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The standard solution for contained (in mm): 5 × TG, 2 MgCl2, 5 1 and 10 Hepes (pH with solution was with CaCl2 and EGTA to buffer or with 10 mm EGTA to buffer The solution contained (in NaCl, KCl, CaCl2, MgCl2, d-glucose, and Hepes (pH 7.4 with NaOH). of was as described previously (27Quintana A. Hoth M. Cell Calcium. 2004; 36: 99-109Crossref PubMed Scopus (20) Google Scholar) with mAb or anti-human Cells were with and and until min and were and are as = of cells. In data were a or was used. could not be a was of are in 1, 4 and 5 to to and to influx through CRAC channels the translocation of mitochondria to the plasma of the mitochondrial localization to the plasma membrane between the in at of Ca2+ influx by 1 as shown in the of the mitochondrial localization to the plasma membrane and at the of the Ca2+ with (in μm) 1 in in 1 and mm Ca2+ Ringer's solution. of the mitochondrial localization to the plasma membrane at the of the Ca2+ and min removing Ca2+ solution in cells. of the mitochondrial localization to the plasma membrane the cells for 30 min with (in μm) 50 and 100 of the mitochondrial localization to the plasma membrane in the of 100 nm BTP2 store and at the of the Ca2+ in cells. confocal and of mitochondrial Confocal from Jurkat T-cells before and Ca2+ influx by the of 1 mm Ca2+ Ringer's solution in or cells. The of the of mitochondria in a from the plasma membrane is at the were and the cells. For cells were with and mouse anti-human CD45-Alexa Fluor®488 mAb the plasma The of the of mitochondria in a nm from the plasma membrane is at the were cells for translocation the plasma membrane along microtubules is required to sustain Ca2+ signal in T-cells. experiments as shown in 1, that cells were with 2 for min in A and or in and min of min of or min of of the mitochondrial localization to the plasma membrane in T-cells and min with 1 mm Ca2+ Ringer's solution 1 TG, as shown in F. were cells cells cells of T-cells for min or not with 2 MitoTracker® of CRAC channel activity on mitochondrial movement the plasma of CRAC channel at are shown with mm mm EGTA and or 10 mm EGTA and in the solution. of the CRAC min the as a of the for the shown in motor proteins control mitochondrial translocation the plasma membrane and sustained Ca2+ signals. MitoTracker® and from MitoTracker® or T-cells before and with 1 in 1 mm Ca2+ Ringer's solution. MitoTracker® and were before and at the of the Ca2+ cell of the mitochondrial localization to the plasma membrane in T-cells = at the of the Ca2+ in T-cells = and at the of the in T-cells = experiments as the in that cells were with mAbs as the control or The at was for mAbs and mAb cells. MitoTracker® Ca2+ Mitochondrial the the of or not directed mitochondrial movement was important for Ca2+ signaling and Ca2+-dependent T-cell activation, we used to the intracellular localization of CRAC channels are by Ca2+ store depletion and by store (2Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1788) Google Scholar), we T-cells with TG, which has been shown to CRAC channels by the Ca2+ A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The intracellular localization of mitochondria was before and T-cell by the of in the and in the of Ca2+, a standard to Ca2+ influx through CRAC channels that sustained Ca2+ influx was with mitochondrial translocation the plasma is the in a membrane MitoTracker® between the of which reflects the of the plasma membrane, the from the 1, and The with was T-cells were and not their thapsigargin movement the plasma membrane was the sustained Ca2+ cell but not store depletion 500 cell or the of the Ca2+ influx A mitochondrial translocation the plasma membrane was also in the Ca2+ not the mitochondrial by within the cells with a distance of The distance between mitochondria and the plasma membrane not store depletion with the Ca2+ the between the was In the MitoTracker® decreased between the that the mitochondrial the plasma To mitochondrial movement the plasma membrane also in T-cells in the of T-cell we used beads to the cells. a in which the between and cell a rise of the intracellular Ca2+ concentration as previously (27Quintana A. Hoth M. Cell Calcium. 2004; 36: 99-109Crossref PubMed Scopus (20) Google Scholar). In mitochondrial movement the plasma membrane was also as from signals and the of mitochondrial movement were with the cells with In the we included cells that not their following translocation of mitochondria to the plasma membrane in cells that their following Ca2+ depletion not mitochondrial translocation the plasma membrane, we mitochondrial movement in the following experiments by Ca2+ influx through in 1 mm Ca2+ Ringer's as shown in the of A translocation of mitochondria the plasma membrane was 2 min the of Ca2+ the of a Ca2+-dependent mitochondrial movement The of mitochondrial movement on Ca2+ influx was by the between the translocation of mitochondria the plasma membrane and the of the Ca2+ concentration which the for Ca2+ as previously (24Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1995; 131: 655-667Crossref PubMed Scopus (164) Google Scholar). in the of or mm Ca2+, which Ca2+ entry through CRAC channels, a movement of mitochondria the plasma membrane was The movement to be with 1 mm Ca2+ for the of the mitochondrial movement on Ca2+ influx can be from the that mitochondrial translocation the plasma membrane was removing the Ca2+ solution and was abolished in T-cells with the Ca2+ BAPTA Ca2+ influx in T-cells on the activity of CRAC channels (24Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1995; 131: 655-667Crossref PubMed Scopus (164) Google Scholar, S. Prakriya M. Rao A. Lewis R.S. J. 2005; PubMed Scopus Google Scholar), we the intracellular localization of mitochondria in T-cells with the CRAC channel BTP2 C. Strauss B. Schwarz E.C. G. A. Hoth M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). 100 nm BTP2 reduced mitochondrial movement the plasma membrane BTP2 not the which was in the of 100 nm BTP2, Ca2+ entry through the CRAC channel is reduced to a to in which mm are in the solution C. Strauss B. Schwarz E.C. G. A. Hoth M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google and mitochondrial movement the plasma membrane in T-cells following Ca2+ influx through CRAC channels was also by confocal and by In the mitochondrial was translocated to to the plasma membrane the Ca2+ signal the following Ca2+ influx by the of 1 mm Ca2+ solution in an not the of 1 mm Ca2+ solution In we also mitochondrial movement the plasma membrane in T-cells with mitochondrial protein not The results shown in 1 and 2 that the directed movement of mitochondria the plasma membrane on Ca2+ influx through CRAC channels in T-cells. Mitochondria the for Sustained Ca2+ mitochondrial movement the plasma membrane following the activation of Ca2+ influx through CRAC channels was abolished by of the cells with a A and but with a not The of mitochondrial movement the plasma membrane by reduced the sustained Ca2+ with control and The of microtubules by is the of membrane MitoTracker® between the as as the sustained Ca2+ signal with the and The Ca2+ Ca2+ influx the of the plasma membrane Ca2+ the of the intracellular Ca2+ concentration Hoth M. Lewis R.S. J. Physiol. (Lond.). 2002; Scopus Google Scholar). To this we also the of to control by Ca2+ influx in the of mitochondrial movement the plasma membrane was abolished by and the rise was also reduced not with mitochondrial Ca2+ or plasma membrane Ca2+ activity that the sustained which on Ca2+ influx through CRAC channels, is through mitochondrial movement the plasma A of this is that of mitochondrial movement into the of the plasma membrane CRAC Mitochondrial the CRAC CRAC channel we used the of the CRAC channels were activated through depletion of the Ca2+ store a of and In a of we CRAC channel inactivation a concentration of the Ca2+ EGTA in the which is not to Ca2+-dependent inactivation of CRAC channels M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (234) Google Scholar). mitochondrial Ca2+ to the channels is required to CRAC channel activity by of Ca2+ that Ca2+-dependent CRAC channel inactivation also 16Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (234) Google Scholar). of T-cells with the movement of mitochondria along microtubules the plasma membrane the Ca2+ influx through CRAC channels, our that CRAC be a inactivation of CRAC channels in T-cells with control and To that this is not a of on the CRAC channels or an we the experiments in the of 10 mm EGTA in the which is to buffer Ca2+, thereby preventing CRAC channel on CRAC channel inactivation and in mitochondrial Ca2+ to the plasma membrane is not required to Ca2+-dependent CRAC channel inactivation in the of 10 mm EGTA we can that of were for the inactivation of CRAC in The of CRAC inactivation is with the of mitochondrial movement the plasma membrane within the 2 min following activation of Ca2+ influx the experiments in and we that mitochondrial movement the plasma membrane is required to CRAC channel activity and sustained signals. Mitochondrial the and Sustained Ca2+ motor proteins control the movement of and along microtubules Cell. 2003; 112: Full Text Full Text PDF PubMed Scopus Google Scholar). the translocation of mitochondria the plasma membrane is an we mAb into T-cells and mitochondrial translocation and signals following mAb the mitochondrial translocation the plasma membrane in with control with anti-human and this movement with a of the sustained Ca2+ signal the importance of mitochondrial localization plasma membrane for Ca2+ signals and Ca2+-dependent signal transduction in T-cells. The of the mAb a of the of the Ca2+ signal Ca2+ which was not in cells with that this may be to the that mitochondria to the the function of has been A. Johnson-Cadwell L.I. Cirulli V. Yoon Y. Allan V.J. Rutter G.A. J. Cell Sci. 2004; 117: 4389-4400Crossref PubMed Scopus (192) Google Scholar, A. Cirulli V. Rutter G.A. Cell Calcium. 2004; 36: PubMed Scopus Google Scholar, R. Nat. Rev. 2005; 6: PubMed Scopus Google Scholar), may the of The of mitochondria to the has been shown to reduce Ca2+ signals A. Cirulli V. Rutter G.A. Cell Calcium. 2004; 36: PubMed Scopus Google Scholar). The data with the mAb the translocation of mitochondria the plasma membrane following the Ca2+ rise in motor proteins along microtubules by a that of A. M. Science. 2004; PubMed Scopus Google Scholar). have shown that of Ca2+ entry in T-cells translocation of the mitochondrial the plasma membrane in a along microtubules Ca2+ influx through CRAC channels directed mitochondrial The of the directed unknown. The mitochondrial movement the plasma membrane in CRAC channel activity and by Ca2+-dependent CRAC channel to this as a that is by CRAC channel activation through depletion of Ca2+ Ca2+ influx through activated CRAC channels mitochondrial translocation to the plasma membrane, which in CRAC channels are of such for which Ca2+, which to which in to a by Hajnoczky and (20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar), the submembranous (in our through CRAC mitochondrial thereby them to the plasma between the of Hajnoczky and (20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar) and our Hajnoczky and show that mitochondrial is reduced in a cell Ca2+ and by the Ca2+ signal a They that mitochondria are to microdomains of Ca2+ the In we not such a Ca2+ and T-cell activation is determined by Ca2+ influx through CRAC channels. Ca2+ in T-cells is in large the probably be for mitochondria in the Ca2+-dependent mitochondria movement the be to such movement not from random In the is a for a Ca2+-dependent the plasma membrane, be by the microdomains to CRAC channels. the data by Hajnoczky and (20Yi M. Weaver D. Hajnoczky G. J. Cell Biol. 2004; 167: 661-672Crossref PubMed Scopus (378) Google Scholar) and our data may be an movement to Ca2+ signals may not be from random movement in the is the Ca2+ the may mitochondria (in our at the plasma In in such as may be directed movement of mitochondria to the plasma membrane, mitochondria are to the influx channel (in Ca2+ influx through CRAC channels the of mitochondria to the plasma membrane and this localization CRAC channels CRAC we that CRAC channels and mitochondria control in a The in the transcription factor from to a of NFAT, and also 3Quintana A. Griesemer D. Schwarz E.C. Hoth M. Pfluegers Arch. 2005; 450: 1-12Crossref PubMed Scopus (89) Google and F. Garcia-Cozar F. Im S.H. Horton H.F. Byrne M.C. Rao A. Cell. 2002; 109: 719-731Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). this to a protein a in mitochondrial localization is into a large on T-cell with