Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m312366200
ISSN1083-351X
AutoresMaud Frieden, Dominic I. James, Cyril Castelbou, Anne Danckaert, Jean‐Claude Martinou, Nicolas Demaurex,
Tópico(s)Metabolism and Genetic Disorders
ResumoMitochondria modulate Ca2+ signals by taking up, buffering, and releasing Ca2+ at key locations near Ca2+ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca2+ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca2+ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca2+ entering across plasma membrane channels, because the Ca2+ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca2+ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by ∼35%. These observations indicate that close contact to Ca2+ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca2+ propagation within interconnected mitochondria. Mitochondria modulate Ca2+ signals by taking up, buffering, and releasing Ca2+ at key locations near Ca2+ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca2+ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca2+ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca2+ entering across plasma membrane channels, because the Ca2+ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca2+ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by ∼35%. These observations indicate that close contact to Ca2+ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca2+ propagation within interconnected mitochondria. Mitochondria actively participate to the cellular Ca2+ homeostasis and modulate the pattern of agonist-induced Ca2+ signals by their ability to sequester and release Ca2+ (1Duchen M.R. J. Physiol. 1999; 516: 1-17Crossref PubMed Scopus (527) Google Scholar). Because of the low Ca2+ affinity of the uniporter that constitutes the main mechanism of Ca2+ entry into mitochondria, it was proposed that the ability of these organelles to accumulate Ca2+ relies on their close location to Ca2+ release channels on the endoplasmic reticulum (ER) 1The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; CCCP, carbonylcyanide m-chlorophenylhydrazone; [Ca2+]mit, mitochondrial [Ca2+]; [Ca2+]cyt, cytosolic [Ca2+]; RP3.1mit, ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; ICRAC, Ca2+ release-activated Ca2+ current; Δψm, mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein. 1The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; CCCP, carbonylcyanide m-chlorophenylhydrazone; [Ca2+]mit, mitochondrial [Ca2+]; [Ca2+]cyt, cytosolic [Ca2+]; RP3.1mit, ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; ICRAC, Ca2+ release-activated Ca2+ current; Δψm, mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein. (2Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (985) Google Scholar, 3Rizzuto 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). Mitochondria also interact with plasma membrane channels and thereby modulate the so-called capacitative Ca2+ entry (CCE) pathway, the ubiquitous Ca2+ entry mechanism triggered by emptying of the ER Ca2+ store (4Putney J.J. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2077) Google Scholar, 5Putney J.J. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1255) Google Scholar). Although the molecular identity of the channel(s) responsible for CCE as well as its mechanism of activation are still debated, recent evidence indicates that mitochondria represent a key organelle in CCE activity and/or activation. Indeed, CCE is inhibited by intracellular Ca2+ elevations, and mitochondria were shown to act as local buffers to prevent Ca2+-mediated inhibition of the CCE pathway (6Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (459) Google Scholar, 7Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (229) Google Scholar, 8Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Malli R. Frieden M. Osibow K. Graier W.F. J. Biol. Chem. 2003; 278: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Local interactions between mitochondria and other subcellular structures are difficult to establish in living cells because mitochondria display a complex architecture that varies considerably between cell types. This ranges from a largely interconnected tubular network in COS-7, endothelial, or HeLa cells to round punctuated structures in hepatocytes (10Collins T.J. Berridge M.J. Lipp P. Bootman M.D. EMBO J. 2002; 21: 1616-1627Crossref PubMed Scopus (455) Google Scholar). Moreover, mitochondria are highly dynamic organelles that move in the cytosol and that constantly undergo fusion and fission. Both processes are under the control of certain GTPases and their associated proteins (11Shaw J.M. Nunnari J. Trends Cell Biol. 2002; 12: 178-184Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). hFis1, the human orthologue of the yeast Fis1p (12Mozdy A.D. McCaffery J.M. Shaw J.M. J. Cell Biol. 2000; 151: 367-380Crossref PubMed Scopus (523) Google Scholar), is a 17-kDa transmembrane protein located in the outer membrane of the mitochondria that is involved in the machinery of mitochondria fission, and overexpression of this protein enhances the fission process in HeLa cells (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). In this study, we overexpressed the protein hFis1 in HeLa cells to induce a controlled fragmentation of mitochondria and measured the impact of these structural changes on cytosolic and mitochondria Ca2+ signals with fura-2 and with a targeted ratiometric pericam probe, respectively. This approach allowed us to investigate the role of mitochondria interconnection on cytosolic and mitochondrial Ca2+ homeostasis and to distinguish the local and global effects of mitochondria on the Ca2+ entry process. Materials—Minimum essential medium, fetal calf serum, penicillin, and streptomycin were obtained from Invitrogen. Histamine, thapsigargin, oligomycin, and rotenone were obtained from Sigma. Acetoxymethyl ester form of fura-2 (fura-2/AM) and Mitotracker Red were obtained from Molecular Probes Europe (Leiden, Netherlands). Carbonylcyanide m-chlorophenylhydrazone (CCCP) was obtained from Fluka (Buchs, Switzerland). Transfast transfection reagent was purchased from Promega. Cell Culture and Transfection—HeLa cells were grown in minimum essential medium containing 10% heat-inactivated fetal calf serum, 2 mml-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin and were maintained at 37 °C under 5% CO2. For experiments, cells were plated on 25-mm diameter glass coverslips 2-3 days before use. After reaching 40-60% of confluence, cells were transiently transfected with the different plasmids using the Transfast reagent according to the protocol supplied by the manufacturer. For measurements of cytosolic Ca2+ concentration, [Ca2+]cyt, hFis1 was co-transfected with a GFP targeted to the nucleus to identify cells expressing hFis1. All experiments were performed 16-20 h after transfection with hFis1. To measure mitochondrial Ca2+, [Ca2+]mit, cells were transfected with the ratiometric pericam targeted to the mitochondrial matrix (RP3.1mit, a gift from Dr. Atsushi Miyawaki, RIKEN Brain Science Institute, Wako-city, Japan) and 24 h later with hFis1. Organelle Imaging and Morphometric Analysis—Organelle morphology was imaged on an Axiovert 200M equipped with an array laser confocal spinning wheel (Nipkow disc; Visitech, Sunderland, UK) using a ×63, 1.4 NA oil-immersion objective (Carl Zeiss AG, Feldbach, Switzerland). Images were acquired on a cooled, 16-bit CCD camera (CoolSnap HQ; Roper Scientific, Trenton, NJ) operated by the Metamorph 5.0 software (Universal Imaging, West Chester, PA). Images shown in Figs. 1A and 3 were deconvolved with the Huygens algorithm (Scientific Volumetric Imaging, Ilversum, The Netherlands) using the Imaris software (Bitplane AG, Zurich, Switzerland). To determine the surface of the cytosol occupied by mitochondria, HeLa cells stably expressing a cytosolic pericam probe were loaded with Mitotracker Red (500 nm for 90 s) to label mitochondria. The cytosolic and mitochondrial stainings were imaged using 488 nm excitation, 535 nm emission, and 546 nm excitation, >580 nm emission, respectively. Optical slices of 200 nm step size in z section were acquired. Of the stack, the five most informative images were visually selected. This corresponded for all cells (control and transfected) to five adjacent slices located at the bottom of the cells. Following this selection, a series of filters was applied to each image before performing mitochondria and membrane segmentation. First, an "autodensity filter" was applied to increase the contrast, followed by an "inverse video" of the images. An automatic threshold corresponding to the histogram average was applied to generate binary images, and a "median filter" was used to smooth the relevant information. The borders of mitochondria and the membrane were segmented from the filtered signal automatically. For the mitochondrial segmentation, objects smaller than a perimeter of 20 pixels were not taken into account. The border coordinates were exported in an Excel file (points x, y), and the impact points between the membrane and the mitochondria borders were calculated. The impact points corresponded to a distance of 0 nm (superimposed pixels) between mitochondrial and membrane borders or <200 nm (neighboring pixels) to match the optical resolution of the confocal images.Fig. 3Mitochondrial pH and Ca2+uptake is not altered by the fragmentation of the mitochondrial network. HeLa cells were transiently transfected with the RP3.1mit, and the pH and Ca2+ sensitivity of the probe are demonstrated in panels A and B. A, 1 μm CCCP was applied to acidify the mitochondrial matrix. The change in pH is reflected by a drop of the fluorescence following excitation at 480 nm. The 410-nm wavelength showed almost no change. B, the cell was stimulated with histamine that induced a mitochondrial Ca2+ uptake, which is reflected by a decrease of the 410-nm excitation wavelength, whereas the 480 nm only marginally changed. C-F, HeLa cells were transfected with the RP3.1mit and 24 h later with hFis1. C, the application of 1 μm CCCP, which dissipated the H+ gradient across the mitochondrial matrix, produced a similar change of the fluorescence recorded after excitation at 480 nm. E, statistical evaluation of the effect of CCCP. Bars are mean ± S.E. (n = 12 for the untransfected cells and 6 for the hFis1-overexpressing cells). D, representative recording of the mitochondrial Ca2+ increase following stimulation with 50 μm histamine in Ca2+-free medium. F, statistics of the amplitude of Ca2+ increase induced by histamine. Bars are mean ± S.E. (n = 33 for the untransfected cells and 13 for the hFis1-overexpressing cells).View Large Image Figure ViewerDownload (PPT) Cytosolic Ca2+Measurements—Experiments were performed in Hepes-buffered solution containing (in mm): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 20 Hepes, 10 glucose, pH 7.4 with NaOH. Ca2+-free solution contained 1 mm EGTA instead of CaCl2, except for the Mn2+ quench experiments, where no EGTA was added. Glass coverslips were mounted in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and vacuum outlet for solution changes. Cells were imaged on a Axiovert s100 TV using a ×100, 1.3 NA oil-immersion objective (Carl Zeiss AG, Feldbach, Switzerland). HeLa cells were loaded for 30 min with 2 μm fura-2/AM at room temperature in the dark, washed twice, and equilibrated for 15-20 min to allow de-esterification. To monitor [Ca2+]cyt, cells were alternatively excited at 340 and 380 nm with a monochromator (DeltaRam; Photon Technology International Inc., Monmouth Junction, NJ) through a 430 DCLP dichroic mirror. Emission was monitored through a 510WB40 filter (Omega Optical, Brattleboro, VT). Prior to the experiments, cells were loaded with 500 nm Mitotracker Red for 90 s and washed two to three times with experimental buffer. Transfected cells were recognized by the fluorescence of the nuclear-targeted GFP (480 nm excitation, 535 nm emission), and the characteristic morphology of mitochondria (fragmented versus tubular) was verified by imaging the mitotracker labeling (577 nm excitation, 590 nm emission). The fluorescence of the nuclear GFP was also observed following excitation at 380 nm during the measurement of [Ca2+]cyt, especially if the labeling was strong. For this reason, we only selected cells with a moderate nuclear fluorescence, and the region of the cytosol used to estimate [Ca2+]cyt did not include the nucleus. Fluorescence emission was imaged using a cooled, 16-bit CCD back-illuminated frame transfer MicroMax camera (Princeton Instruments, Ropper Scientific, Trenton, NJ). Image acquisition and analysis were performed with the Metafluor 4.6 software (Universal Imaging, West Chester, PA). Measurements of Store-operated Ca2+Entry—Mn2+ (100 μm) was substituted for Ca2+ to estimate the ion flux through store-operated Ca2+ channels, according to the Mn2+ quench technique. Cells were excited at 356-358 nm, which corresponded to the isosbestic point of fura-2. The rate of fluorescence decrease reflects the rate at which Mn2+ enters the cells, and the slope during the first 1-2 min was used as an indicator for CCE activity. Mitochondrial Membrane Potential (Δψm) Measurements—To monitor changes in Δψm, cells were loaded for 20 min with 50 nm tetramethylrhodamine methyl ester (TMRM) in Hepes-buffered solution, and experiments were carried out in the same buffer. Cells were excited at 545 nm and emission collected through an LP 590 long pass filter. Changes in Δψm were expressed as R/Ro, where R is the ratio of the fluorescence in the mitochondria divided by the cytosolic fluorescence at a given time and Ro is the initial ratio of the mitochondrial over cytosolic fluorescence. Mitochondrial Ca2+and pH Measurements—We took advantage of the properties of RP3.1mit, whose fluorescence is Ca2+-sensitive when excited at 410 nm and pH-sensitive when excited at 480 nm (Refs. 14Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar, 15Filippin L. Magalhaes P.J. Di Benedetto G. Colella M. Pozzan T. J. Biol. Chem. 2003; 278: 39224-39234Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, see also Fig. 3). The cells were excited alternatively at 410 and 480 nm, and emission was collected at 535 nm (535RDF45; Omega Optical) through a 505DCXR (Omega Optical) dichroic mirror. Changes in pH were expressed as F/Fo, where F is the fluorescence (480 nm excitation) at a given time and Fo is the mean fluorescence of 5-10 individual measurements collected at the beginning of the recording. Changes in mitochondrial Ca2+ are shown as 1-F/Fo, because RP3.1mit fluorescence at λexc = 410 nm decreases with increasing Ca2+ concentrations. Effects of hFis1 Expression on Mitochondria Morphology and Location—Mitochondria in HeLa cells form a largely interconnected network constantly remodeled by fusion and fission reactions. To disrupt this balance toward fission, we overexpressed hFis1, the human orthologue of the yeast protein Fis1p known to participate in mitochondrial division. As shown in Fig. 1A, expression of hFis1 rapidly fragmented the mitochondrial network into punctuate organelles that clustered around the nucleus. The fragmentation process occurred immediately upon hFis1 expression and, once initiated, was complete within 4 h as documented by time-lapse video microscopy of cells co-transfected with a nuclear-targeted GFP (See supplementary movie S1). Within 1 h, all the cells expressing the hFis1 cDNA had a punctiform mitochondria phenotype, consistent with a previous study (13James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). To quantify the extent of mitochondrial redistribution, we took several confocal optical sections of mitochondria (labeled with Mitotracker Red) and of the cell cytosol (labeled with the cytosolic protein ratiometric pericam). Using the mitochondrial image as a mask, we determined the surface of the cell occupied by mitochondria on the cytosolic images (the nucleus was included in the cell surface, see "Experimental Procedures"). As shown in Fig. 1B, mitochondria spread out to the periphery and covered a larger area of the cytosol in control cells. On average, the percentage of the cell surface "lacking" mitochondria was ∼2-fold larger in hFis1-expressing cells compared with control (Fig. 1C, n = 15 and 32 cells, respectively). The same images were used to measure the number of contact points between mitochondria and the cell membrane, defined as the outline of the cytosolic staining. As shown in Fig. 1D, ∼18% of the cell membrane was apposed to mitochondria in control cells (at the resolution of our confocal system of <250 nm), a proportion that was reduced by ∼3 times upon hFis1 expression. Thus, hFis1 not only induces mitochondrial fragmentation but also redistributes mitochondria away from the plasma membrane, leaving large regions of the cell periphery devoid of mitochondria and fewer contacts between mitochondria and the cell surface. Effects of hFis1 Expression on Mitochondrial Membrane Potential, pH, and Ca2+Homeostasis—To assess the effects of hFis1 expression on mitochondrial function, we first tested whether the mitochondrial membrane potential (ΔΨm) was altered after fragmentation. For this purpose, cells were loaded with TMRM and challenged sequentially with 5 μg/ml oligomycin (to prevent ATP consumption) and 25 μm rotenone (to block the complex I of the respiratory chain). As shown in Fig. 2, the drugs elicited similar changes in ΔΨm in hFis1-overexpressing and control cells. In both cases, neither the application of oligomycin nor rotenone alone had a significant effect on ΔΨm, whereas their combined application dissipated ΔΨm to a similar extent (Fig. 2B). The subsequent addition of the protonophore CCCP induced a rapid and complete dissipation of the membrane potential. The lack of depolarization in the presence of oligomycin indicates that the respiratory chain was functional in hFis1 cells and that the mitochondrial membrane potential was not maintained by the mitochondrial H+ ATPase functioning in reverse mode and consuming glycolytic ATP. Next, we measured the H+ and Ca2+ activities inside the mitochondrial matrix of live HeLa cells. For these measurements, we took advantage of the dual sensitivity to both Ca2+ and pH of a ratiometric pericam probe targeted to mitochondria, RP3.1mit (kindly provided by Dr. A. Miyawaki, Tokyo). RP3.1mit fluorescence is highly sensitive to pH at an excitation of 480 nm, but not at 410 nm (14Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar, 15Filippin L. Magalhaes P.J. Di Benedetto G. Colella M. Pozzan T. J. Biol. Chem. 2003; 278: 39224-39234Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Conversely, RP3.1mit fluorescence decreases with increasing concentrations of Ca2+ at 410 nm but is largely insensitive to Ca2+ at 480 nm. We could verify this dual sensitivity by exposing RP3.1mit-labeled HeLa cells to the mitochondrial uncoupler CCCP or to the calcium-mobilizing agonist histamine. As expected, CCCP caused a selective drop in RP3.1mit fluorescence at 480 nm as the mitochondria acidified to equilibrate its pH with the pH of the cytosol (Fig. 3A). In contrast, addition of histamine produced a transient drop only at 410 nm (Fig. 3B), confirming that changes in mitochondrial Ca2+ concentration, [Ca2+]mit, could be monitored selectively at this wavelength. We used this approach to evaluate the effect of hFis1 expression on mitochondrial Ca2+ and pH homeostasis. As shown in Fig. 3, C and E, addition of 1 μm CCCP elicited a drop in fluorescence at 480 nm that was of similar magnitude in control and in hFis1-expressing cells. The drop in fluorescence corresponded to a similar ΔpH, because the RP3.1mit calibration curves were similar in hFis1-overexpressing and -untransfected cells in the pH range 7.4-8.4 (see Supplementary Fig. 2). These data confirm the TMRM measurements and indicate that the pH of the mitochondrial matrix was not altered by hFis1 expression. In concurrent Ca2+ measurements at 410 nm, addition of 50 μm histamine evoked identical [Ca2+]mit transients regardless of the induction of hFis1 (Fig. 3D). In the absence of extracellular Ca2+, neither the maximal [Ca2+]mit elevation nor the duration of the response was different in control and hFis1-overexpressing cells (Fig. 3F). The [Ca2+]mit transients measured in the presence of extracellular Ca2+ were also not significantly different; the maximal amplitude of the signal averaged 0.167 ± 0.015 (n = 33) in untransfected cells and 0.141 ± 0.015 (n = 13) in hFis1 overexpressors. Taken together, these data indicate that mitochondrial pH and Ca2+ homeostasis is not affected by hFis1 expression despite the complete fragmentation of the mitochondrial network. Effects of hFis1 Expression on ER Structure—The presence of normal [Ca2+]mit transients in HeLa cells with fragmented and clustered mitochondria is surprising, because the proximity of mitochondria to the Ca2+ source (i.e. the inositol 1,4,5-trisphosphate Ca2+ release channels) was shown to be crucial for a proper mitochondrial Ca2+ uptake. Mitochondria have been proposed to form stable, long-term interactions with ER Ca2+ release channels to account for the efficient transfer of Ca2+ between the two organelles (15Filippin L. Magalhaes P.J. Di Benedetto G. Colella M. Pozzan T. J. Biol. Chem. 2003; 278: 39224-39234Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Because hFis1 induced dramatic alterations in mitochondrial architecture, we investigated whether the ER was also affected. As shown in Fig. 4, the staining pattern of the ER-targeted yellow cameleon probe (YC4.1ER) was not grossly altered upon hFis1 expression, indicating that the mitochondrial remodeling was not accompanied by visible changes in ER architecture. Effects of hFis1 on Ca2+Transfer from Plasma Membrane Channels to Mitochondria—hFis1-fragmented mitochondria appear to handle normally the Ca2+ released from the ER, as indicated by the normal [Ca2+]mit transient elicited by histamine. Because the main effect of hFis1, apart from fragmentation, is to move mitochondria away from the plasma membrane (Fig. 1), we assessed whether hFis1 altered the ability of mitochondria to take up Ca2+ originating from the plasma membrane. For this purpose, Ca2+ was readmitted to cells previously stimulated with 50 μm histamine in the nominal absence of Ca2+. As shown in Fig. 5A, the amplitudes of the [Ca2+]mit elevations were similar in control (0.109 ± 0.019; n = 12) and in hFis1-overexpressing cells (0.110 ± 0.014; n = 15). Interestingly however, the time needed to reach this level was significantly prolonged by hFis1 expression. The Ca2+ entering across the plasma membrane took, on average, 31 s longer to cause a maximal response in hFis1-fragmented mitochondria. Similar results were obtained in cells stimulated with the SERCA pump inhibitor, thapsigargin (1 μm), instead of histamine (Fig. 5, C and D), indicating that ER Ca2+ pumps were not involved in the transfer of Ca2+ from the plasma membrane to mitochondria. To understand the structural basis of this slower [Ca2+]mit increase, we analyzed the spatio-temporal pattern of the [Ca2+]mit signal during Ca2+ readdition to control and hFis1 cells. As shown in Fig. 6, [Ca2+]mit increased rapidly in large, contiguous regions of control tubular mitochondria. In contrast, [Ca2+]mit increased sequentially in discrete regions of hFis1-fragmented mitochondria. The slower response of hFis1-fragmented mitochondria was not because of a delay in the transfer of Ca2+ from the plasma membrane to mitochondria, because the [Ca2+]mit signal initiated at the same time or even earlier in individual mitochondria from hFis1-transfected cells (Fig. 6A). Rather, Ca2+ spread faster and in a more coordinated manner within tubular mitochondria (Fig. 6B), indicating that the propagation of the [Ca2+]mit signal was impaired by the fragmentation of the mitochondrial network. The delayed [Ca2+]mit increase in hFis1 cells might possibly reflect a reduced or slower influx of Ca2+ across the plasma membrane. To test this possibility, we measured Ca2+ influx with fura-2. As shown in Fig. 7, the cytosolic Ca2+ changes upon Ca2+ readdition to cells stimulated with thapsigargin were of similar amplitude and kinetics in control and hFis1-overexpressing cells. To confirm this observation, CCE activity was measured by following the rate of Mn2+ influx (Fig. 7C). The rates of Mn2+ quench were not significantly different in control and hFis1-expressing cells (Fig. 7D), indicating that CCE was largely unaffected by the fragmentation and subcellular redistribution of the mitochondrial network. Effects of hFis1 on CCE Modulation by Mitochondria—Functional mitochondria are required to sustain CCE, but it is not clear whether mitochondria act locally, i.e. as Ca2+ buffers that remove Ca2+-dependent channel inhibition, or globally, i.e. by modulating the filling state of the ER or by releasing a diffusible messenger. Because mitochondria in hFis1 cells were clearly located farther away from the plasma membrane than in untransfected cells, they provided a convenient model to separate the local and global effects of mitochondria on CCE. For this purpose, cells were stimulated with thapsigargin to activate CCE and mitochondria function was inhibited by either 1 μm CCCP or by a combination of 25 μm rotenone and 5 μg/ml oligomycin. The effects of the mitochondria inhibitors on CCE were then assessed by the Ca2+ readdition protocol or by the Mn2+ quench technique. As shown in Fig. 8, Ca2+ entry was reduced by about one third in the presence of CCCP or of oligomycin/rotenone, regardless of hFis1 expression. Mn2+ entry was reduced to a similar extent in the presence of 1 μm CCCP, both in control (from 8.503 ± 1.442, n = 18 to 4.630 ± 0.464, n = 25; p <0.05) and in hFis1-overexpressing cells (from 6.876 ± 1.196, n = 14 to 3.276 ± 0.225, n = 10; p <0.05). These results indicate that functional mitochondria are required for optimal activation of CCE in HeLa cells, although the modulation of CCE by mitochondria (30-40%) is less pronounced than in other cell types. In this study we investigated the effect of a controlled disruption of the mitochondrial network on the Ca2+ homeostasis of mitochondria. For this purpose, we expressed the protein hFis1 in HeLa cells to induce a rapid fragmentation and perinuclear clustering of their mitochondria. Surprisingly, these dramatic morphological alterations had little impact on the organelle function because mitochondria were still able to m
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