Báo cáo khoa học: Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation pdf

Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation Angela M. Falchi, Raffaella Isola, Andrea Diana, Martina Putzolu and Giacomo Diaz Department of Cytomorphology, University of Cagliari, Monserrato, Italy Fluctuations of the mitochondrial membrane potential (MMPFs) have been investigated in mitochondria of intact cells [1–7] and in isolated mitochondria [2,8,9] stained with tetramethylrhodamine derivatives (TMRM, TMRE and related compounds, hereafter indicated as TMRM). It has been postulated that mitochondrial depolarization is due to singlet oxygen generated by the photoactivation of TMRM [10]. Depo- larization is followed by the efflux of the fluorescent probe, which stops the production of singlet oxygen. This allows the mitochondrial potential to be recovered, followed by a new influx of TMRM from the cytosol. Thus, the continuous illumination of TMRM- stained mitochondria triggers cyclic depolarization and repolarization phases, at least as long as mitochondria are able to counterbalance the oxidative and dissipative effects. A hypothetical model of MMPFs induced by TMRM photoactivation is shown in Fig. 1. If the role of TMRM is evident, on the other hand, the mechanism directly responsible for the mitochond- rial depolarization is not clear. The existence of a link between NAD(P)H, reactive oxygen species (ROS) and the permeability transition pore (PTP) has been dem- onstrated in numerous studies. However, the complex- ity of the interactions between ROS, NAD(P)H, PTP and mitochondrial potential does not allow the majority of phenomena to be represented by a simple cause–effect relationship. For example, ROS cause Keywords fluorescent probes; mitochondria; photoactivation; potential fluctuations; tetramethylrhodamine methyl ester (TMRM) Correspondence G. Diaz, Department of Cytomorphology, University of Cagliari, I-40492 Monserrato, Italy Fax: +39 70 6754003 Tel: +39 70 6754081 E-mail: gdiaz@unica.it (Received 5 October 2004, revised 21 January 2005, accepted 26 January 2005) doi:10.1111/j.1742-4658.2005.04586.x Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were ana- lyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochond- rial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mito- chondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mito- chondria accompanied by NAD(P)H oxidation and Ca 2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o-phenyl-EGTA (a Ca 2+ chelator), suggesting that the permeabil- ity transition pore was not involved in mitochondrial potential fluctuations. Abbreviations CsA, cyclosporin A; DCDHF, 6-carboxy-2¢,7¢-dichlorodihydrofluorescein diacetate diacetomethyl ester; D phase, depolarization phase; IAA, iodoacetic acid; MMPF, mitochondrial membrane potential fluctuation; NP-EGTA, o-nitrophenyl EGTA; PTP, permeability transition pore; R phase, repolarization phase; ROS, reactive oxygen species; SAD, succinic acid dimethyl ester; TMRM, tetramethylrhodamine methyl ester. FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS 1649 depolarization, but they are themselves produced by the respiratory chain at a rate that varies with the membrane potential [11,12]; NAD(P)H energizes the mitochondrion, but NAD(P)H is also an essential sub- strate of glutathione and a direct scavenger of singlet oxygen [10]; PTP opening causes depolarization, but PTP may also be activated by depolarization [13]; PTP-induced depolarization may be inhibited by oxy- gen radical scavengers, catalase and glutathione [2,4,5,14]. Likewise, it is not clear how the mitochondrial potential is restored after the TMRM efflux. Elimin- ation of ROS, if present, and switching of PTP to the close configuration, if previously made to open, are essential but not sufficient conditions for recovery of the mitochondrial potential. Mitochondrial repolariza- tion requires the active support of the respiratory chain and ⁄ or the energetic contribution of ATP hydro- lysis. The latter mechanism has been found to occur in response to depolarization induced by protonophores or Ca 2+ overloading [15,16]. Moreover, ATP hydro- lysis is the sole mechanism capable of energizing DNA-depleted, metabolically inert mitochondria [17,18], as well as mitochondria of blood eosinophils, which have a functional role in apoptosis but not in respir- ation [19]. The aim of this work was to investigate the mecha- nisms underlying MMPFs induced by TMRM photo- activation, by testing the effects of specific inhibitors on mitochondrial depolarization and repolarization phases, analyzed separately. Results The effect of TMRM photoactivation on the mitoch- ondrial potential can be evaluated by comparing the average curves of mitochondrial depolarization under conditions of continuous and discontinuous illumin- ation (Fig. 2A). However, MMPFs were visible only on plotting data of single mitochondria, and some rep- resentative traces are shown in Figs 2B and 6. The exact identification of depolarization phases (D phases) and repolarization phases (R phases) of MMPFs was obtained by derivative analysis, as illustrated in Fig. 2C–D and Fig. 3 (details are given in Experimen- tal Procedures). Generation of ROS by mitochondria exposed to TMRM illumination was detected by 6-carboxy-2¢,7¢- dichlorodihydrofluorescein diacetate diacetomethyl ester (DCDHF) (Fig. 4). ROS were not observed in mitochondria exposed to light in the absence of TMRM, nor in mitochondria filled with TMRM but not exposed to light [20]. The intensity of ROS, as detected by DCDHF, was roughly proportional to the TMRM fluorescence. This confirmed the necessity of selecting a homogeneous baseline fluorescence intensity in order to avoid experimental data being confounded by the effect of the initial amount of TMRM accumu- lated in mitochondria [21]. In all experimental groups, the frequency of D phases prevailed over that of R phases, resulting in a net depolarization at the end of the illumination per- iod. In untreated cells, the ratio between D and R phases was about 3 : 1. The frequency of R phases was significantly reduced by rotenone, oligomycin, auro- vertin, and iodoacetic acid (IAA) (Fig. 5A). The effect of azide (P ¼ 0.07) was not significant but close to the critical threshold. R phases were almost completely abolished by the combination of aurovertin plus olygo- mycin, and rotenone plus olygomycin. The effect of rotenone was removed by combination with succinic acid dimethyl ester (SAD). These findings suggest that the activity of both respiratory chain and ATP syn- thase is required to activate the R phase. On the other hand, the frequency of D phases was substantially stable. The frequency of D phases was not affected by Fig. 1. Schematic model of MMPFs induced by TMRM photoactiva- tion under continuous illumination conditions. TMRM photoactiva- tion results in the generation of singlet oxygen and NAD(P)H oxidation. Possible intermediary effectors of depolarization (super- oxide, Ca 2+ , permeability transition pore, inner membrane anionic channels, etc.) are indicated by the ‘?’ symbol. Depolarization is fol- lowed by the efflux of TMRM, which interrupts the generation of singlet oxygen and allows the mitochondrial potential to be recov- ered by the respiratory chain. Repolarized mitochondria accumulate new TMRM which starts a new cycle. Mitochondrial membrane potential fluctuations A. M. Falchi et al. 1650 FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS o-nitrophenyl EGTA (NP-EGTA) and vitamin E, whereas a significant increase was found with cyclospo- rin A (CsA) (Fig. 5B). The Ca 2+ -binding ability of NP-EGTA was verified by the release of Ca 2+ found after NP-EGTA photolysis (data not shown). These findings seem to exclude an involvement of the PTP in the D phase. Accurate analysis of images also excluded the occurrence of mitochondrial swelling, a marker of permeability transition. The duration of D and R phases was 1.05 and 0.77 s, respectively. The rate of fluorescence changes in D and R phases was 15 and 12 gray valuesÆs )1 , respectively (Fig. 5C–D). Interestingly, the rate of R phases was not significantly altered, even when the fre- quency of R phases was extremely low. The sum of all D and R phase changes accounted for 78.4% of the total fluorescence change found at the end of the illumination period (Fig. 2A, curve a). This discrepancy was probably due to the presence of small MMPFs, not distinguishable from noise, which were eliminated by the filtering method. The effect of probe bleaching was negligible (Fig. 2A, curve c). MMPFs were simultaneously detected in all sub- regions of single mitochondrial filaments (Fig. 6). No evidence of longitudinal propagation of MMPFs was ever detected, despite the remarkable length of some mitochondria. However, it cannot be excluded that propagation of MMPFs may actually occur at a speed Fig. 2. TMRM florescence measurements. (A) Effect of continuous (curve a) and discontinuous (curve b) illumination on the mitochond- rial TMRM fluorescence, sampled at time intervals of 1 s. Discon- tinuous illumination consisted of light cycles of 20 ms, sufficient to acquire the image, followed by dark periods of 980 ms. Data repre- sent averages of several cells, so that MMPFs are not visible. Curve c shows the fluorescence decay of TMRM due to photo- bleaching, under continuous illumination. Photobleaching measure- ments were made on dried stains of TMRM to avoid fluorescence recovery after photobleaching. For all measurements, baseline val- ues of fluorescence intensity were in the same range of gray val- ues (70–130). (B) Representative trace of TMRM fluorescence intensity changes occurring in a single mitochondrion, exposed to continuous illumination and sampled at the rate of one image every 60 ms. Typical MMPFs are evident, but the separation of D and R phases is imprecise. (C) Derivative curve of the TMRM trace. R and D phases are readily identified by negative and positive peaks, respectively. (D) Derivative curve after removal of noise (small peaks) and other irregular (asymmetric) fluctuations. The features of noise fluctuations were preliminarily analyzed from the autofluo- rescence of plastic film, using the same optical settings and meth- ods applied to mitochondria. The max height and max width of derivative peaks of noise fluctuations were set as cut-off values for mitochondrial data. The asymmetry of peaks was also considered to exclude irregular fluctuations with nonlinear slopes. Details are given in Experimental Procedures. A. M. Falchi et al. Mitochondrial membrane potential fluctuations FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS 1651 faster than 0.17 lmÆms )1 , which is the detection limit of our system, based on the ratio between the longest mitochondria analyzed (10 lm) and the time interval between consecutive images (60 ms). In contrast with the simultaneous appearance of fluorescence changes, the fluorescence intensity was not uniformly distributed throughout all subregions of single mitochondrial filaments. This fact was initially ascribed to the alternation of in-focus and out-of-focus subregions due to the sinuosity of the mitochondrial filament. This hypothesis was tested by taking images at different focus levels, with the expectation of obser- ving a progressive shift of fluorescence maxima along the filament. Surprisingly, the longitudinal distribution of fluorescence levels was not modified by focus chan- ges, suggesting that longitudinal differences of fluores- cence intensity were not optical artifacts, but reflected intrinsic properties of mitochondria or of the sur- rounding cytoplasmic environment. TMRM photoactivation resulted in a 34% decrease in the mitochondrial NAD(P)H autofluorescence, in close agreement with other investigations [10,22]. The NAD(P)H decrease after exposure of cells to illumin- ation, in the absence of TMRM, was only 4%. This indicated that TMRM photoactivation was the pri- mary cause of NAD(P)H oxidation. The correlation between TMRM and NAD(P)H changes was detected at the level of single mitochondria (Fig. 7). Unfortu- nately, the relatively long exposure required for NAD(P)H autofluorescence did not allow the occur- rence of NAD(P)H fluctuations in parallel with the acquisition of MMPFs to be verified. Experiments with TMRM and Calcium Green-1 showed a net accumulation of Ca 2+ into mitochondria at the end of the period of TMRM illumination (Fig. 8). The fluorescence change was not attributable to Calcium Green-1 dequenching, consequent on the TMRM efflux, because (a) no quenching of Calcium Green-1 fluorescence was found in TMRM-filled mito- chondria before depolarization, and (b) no dequench- ing was found in TMRM-depleted mitochondria after depolarization induced by fluorocarbonyl cyanide phe- nylhydrazone. No transients of the mitochondrial potential were found after Ca 2+ release induced by ATP stimula- tion of the IP 3 pathway, despite a prominent increase in nuclear and cytoplasmic Ca 2+ followed by synchronous oscillations in both compartments. Ca 2+ oscillations exhibited a constant duration of about 12.5 s, independent of their intensity which was gradually decreasing (see Supplementary mater- ial). In agreement with the inhibition of R phases induced by NP-EGTA, Ca 2+ release resulted in a significant increase in the mitochondrial potential and NAD(P)H content, presumably because of the activation of Ca 2+ -dependent mitochondrial dehydro- genases [23–25]. Discussion MMPFs induced by TMRM photoactivation have been extensively investigated to assess the functional continuity of the mitochondrial network [3–7,26,27]. A less explored aspect of MMPFs concerns the mecha- nisms involved in the cyclic loss and recovery of the mitochondrial potential. In fact, the double role of the fluorescent probe as inducer and detector of MMPFs represents a limitation of experimental studies, as any treatment influencing the baseline TMRM concentra- tion will also modify the generation of MMPFs, thus making it difficult to distinguish effects of different nature. This aspect has not been adequately considered Fig. 3. MMPF recognition. Mitochondrial D and R phases (upper panel) were numerically recognized by derivative analysis as posit- ive and negative peaks (lower panel). For each peak, the features of height, width and symmetry were calculated. The peak height was calculated as the peak amplitude (segment a). The peak width was calculated as the interval between the zero-derivative time points t1 and t2. The peak symmetry was calculated as the lowest of the reciprocal ratios between the t2-p and p-t1 segments. Cut- off values for peak height, width and symmetry were set to filter noise and irregular fluctuations (see Experimental procedures). The change in the original fluorescence intensity scale was obtained as the difference between f1 and f2 values observed at the t1 and t2 time points, respectively. Mitochondrial membrane potential fluctuations A. M. Falchi et al. 1652 FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS in previous studies which examined MMPFs in differ- ent experimental conditions. The effect of singlet oxygen on PTP, and the involvement of PTP in MMPFs induced by photo- dynamic action are still questions open to debate. Contrasting data obtained by the same group of inves- tigators have suggested that PTP may be activated or inhibited by singlet oxygen, in accordance with the nature and localization of the photosensitizer [28,29]. The issue is complicated by the fact that superoxide, a proven PTP inducer, is generated at higher rates by the respiratory chain under oxidative stress conditions, and singlet oxygen may be a substrate for superoxide production at the level of complex III of the respir- atory chain or reacting with NAD(P)H [10]. Further- more, it is not clear whether PTP is actually involved in MMPFs. In some investigations, mitochondrial depolarization induced by TMRM or TMRE photo- activation was inhibited or decreased by CsA [30,31]. In others [2,6,10], the involvement of PTP was exclu- ded. Our data indicate that, at least in HeLa cells, CsA not only does not prevent, but rather increases, the frequency of D phases. In addition, mitochondrial swelling, a classical marker of permeability transition, was never observed during our experiments, even after mitochondria reached a condition of permanent depo- larization. Recently, propagation of MMPFs induced by photo-oxidation has been correlated with the acti- vation of inner membrane anion channels [22,31]. All the above data were obtained in mitochondria of intact cells. An apparent contrast in the behavior of intact cells and isolated mitochondria has been observed by Huser & Blatter [2] who found that depo- larization induced by TMRM photoactivation was pre- vented by CsA in isolated mitochondria but not in mitochondria of intact cells. To explain this discrep- ancy, it was hypothesized that mitochondria of intact cells are less sensitive to CsA because of the abundance of CsA-binding proteins in the cytoplasm. On the other hand, depolarization due to calcium-induced cal- cium release was found by Ichas et al. [32] to be lar- gely prevented by CsA in mitochondria of intact cells. Taken together, these findings indicate that CsA is an effective inhibitor of PTP even in intact cells, but PTP activation in intact cells is more sensitive to calcium stimulation than photoactivation of fluorescent probes. A further difference between these experimental mod- els is the self-propagation of the depolarization that Fig. 4. Generation of ROS by TMRM illumination. Cells were loaded with TMRM and the ROS-sensitive probe DCDHF, with or without pre- incubation with vitamin E. TMRM images (left) were taken at time zero. DCDHF images (right) were taken at the end of the period of TMRM excitation (13.8 s). On comparison of images, a close correspondence is revealed between ROS and mitochondrial traces. However, rather than being confined to the mitochondrial matrix, ROS appear to be spread in the surrounding cytoplasm, thereby suggesting a mech- anism of ROS release. ROS are also present in the nucleus, which is entirely surrounded by the mitochondrial network. Note that the blurred appearance of DCDHF is not an effect of poor focus, and that DCDHF does not accumulate in mitochondria, but is rather uniformly distri- buted throughout the cell [20]. ROS were inhibited by vitamin E. Production of ROS was not elicited by illumination alone in the absence of TMRM, nor, vice versa, by the sole TMRM in the absence of illumination (data not shown). Bar is 10 lm. A. M. Falchi et al. Mitochondrial membrane potential fluctuations FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS 1653 accompanies calcium-induced calcium release [32], whereas depolarization induced by photo-oxidation, at least in the majority of cell types investigated, affects only the irradiated region but does not propagate to adjacent mitochondria [26]. Only in cardiomyocytes, which possess specialized intermitochondrial junctions [26], has a local laser irradiation been found to activate a cell-wide, slow traveling wave of depolarization associated with ROS-induced ROS release [22,31]. However, MMPFs of cardiomyocytes were not preven- ted by intracellular Ca 2+ buffering with EGTA or 1,2-bis-(aminophen oxy)eth ane- N,N,N¢,N¢-tetra-acetic acid (BAPTA), in line with our data on HeLa cells. The increase in mitochondrial Ca 2+ found in our experi- ments after the induction of MMPFs may be explained by local exchanges between mitochondria and neigh- boring Ca 2+ domains of the endoplasmic reticulum [33–36], which may be responsible for the specific behavior of mitochondria of intact cells, as compared with isolated mitochondria. The higher susceptibility of isolated mitochondria to PTP [37] may also be considered in relation to the level of ROS, as isolated mitochondria are generally suppor- ted by succinate ⁄ rotenone, and the rate of ROS gen- eration is much higher in mitochondria respiring using complex II substrates (plus rotenone) than complex I substrates [38]. In addition, MMPFs of isolated mito- chondria supported by NAD(P)H-linked substrates (malate and glutamate) have been found to be insensit- ive to CsA and negative to the calcein assay for PTP opening [9,39]. NAD(P)H is important not only as an energetic sub- strate but also as an antioxidant substrate of glutathi- one and as a singlet oxygen scavenger [10]. However, our data suggest that NAD(P)H has a primary role in respiration, rather than as antioxidant, as R phases, which are more closely correlated to the energetic util- ization of NAD(P)H, were strongly inhibited by rote- none, whereas D phases, which are more closely Fig. 5. Effects of treatments on MMPFs. The four panels show the changes of R and D phase frequencies (number per mitochondrion per minute) and fluorescence change rates (intensity change per second) after treatment with 10 l M NP-EGTA, 50 lM (+)a-toco- pherol acetate (Vit E), 2 l M CsA, 5 lM rotenone plus 7.7 mM SAD (rot + SAD), 6 m M NaN 3 (azide), 50 lM IAA, 5 lM rotenone (rot), 5 l M rotenone plus 10 lM oligomycin (rot + oligo), 10 lM oligomy- cin (oligo), 30 l M aurovertin (auro) and 30 lM aurovertin plus 10 lM oligomycin (auro + oligo). Bars represent the median and interquar- tile range (25th)75th centile). The asymmetry of interquartile ranges is due to the skewness of data distributions. Significant (P<0.05) deviations from controls, calculated by the Student– Newman–Keuls test for multiple comparisons, are indicated by asterisks. Mitochondrial membrane potential fluctuations A. M. Falchi et al. 1654 FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS correlated to oxidative phenomena, were substantially unchanged, in spite of the higher NAD(P)H availabil- ity after complex I inhibition. DCDHF traces were significantly decreased by vita- min E. However, vitamin E failed to inhibit MMPFs. The discrepancy between ROS inhibition and mito- chondrial depolarization may tentatively be explained by the observation that vitamin E is able to reduce ROS present in the cytosol surrounding mitochondria rather than ROS present in the mitochondrial matrix [20]. Contrasting effects of vitamin E have also been found in the inhibition of mitochondrial depolarization of rat and rabbit cardiomyocytes [31]. However, data obtained with specific superoxide scavengers [22] and spin traps [30] have provided consistent evidence that ROS represent a key factor in triggering MMPFs. The average duration of MMPFs (1–2 s) was in close agreement with data obtained in previous studies using relatively fast acquisition methods [2,4,5,8,30,33]. MMPFs of apparently longer duration in literature result from data acquired at lower sampling rates [6,9]. R phases were strongly reduced by IAA, rotenone and ATP synthase inhibitors. Whereas the effect of IAA and rotenone seems to be obvious, that of ATP syn- thase inhibitors is open to different interpretations. One is that ATP synthesis may sustain repolarization by increasing respiration and ⁄ or by regulating the H + influx. A possible alternative is that ATP hydrolysis contributes to respiration to reach a critical H + threshold for the import from the cytosol of energetic substrates. Further investigation of these issues is required. Experimental procedures Cell treatments HeLa cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium with high glucose. Cells were supravitally stained with 100 nm TMRM for 30 min; 18 lm DCDHF Fig. 6. Simultaneous vs. independent MMPFs. Simultaneous MMPFs were found in all subregions of continuous mitochondrial filaments (A). On the other hand, adjacent mitochondria exhibited completely independ- ent MMPFs (B). A. M. Falchi et al. Mitochondrial membrane potential fluctuations FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS 1655 for 60 min; 5 lm Calcium Green-1 AM for 120 min. Cells were treated with 10 lm oligomycin (inhibitor of ATP syn- thase) for 10 min; 30 lm aurovertin (inhibitor of ATP syn- thase) for 30 min; 5 lm rotenone (inhibitor of complex I) for 30 min; 6 mm NaN 3 (inhibitor of complex IV and V) for 30 min; 50 lm IAA (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase) for 30 min; 7.7 mm SAD (substrate of complex II) for 10 min; 2 lm CsA (inhibitor of the PTP) for 30 min; 50 lm (+)a-toco- pherol acetate (vitamin E) for 60 min or overnight; 10 lm NP-EGTA (a cell permeant probe that binds Ca 2+ with high affinity until photolysed by UV light) for 30 min; 20 lm ATP (activator of purinergic receptors and the IP 3 pathway) given at the time of acquisition of images. Drug combinations (aurovertin and oligomycin, rotenone and olygomycin, rotenone and SAD) were used at concentra- tions applied for single treatments. Drug vehicles were: Me 2 SO for TMRM, Calcium Green-1, oligomycin and rote- none; chloroform for aurovertin; ethanol for CsA; water for azide, IAA, vitamin E, NP-EGTA and ATP. Stock solutions were prepared to obtain a 1 : 1000 (0.1%) dilu- tion of vehicles in the medium. TMRM, DCDHF, Calcium Green-1 and NP-EGTA were from Molecular Probes A B Fig. 7. Correlation between TMRM changes and NAD(P)H oxida- tion. (A) NAD(P)H and TMRM images captured at the start and end of the illumination period. Bar is 10 lm. (B) NAD(P)H and TMRM fluorescence of 15 mitochondrial regions of the same cell. Each bar represents the change between the initial (upper edge) and final (lower edge) value. Mitochondrial regions are conventionally ordered according to the final TMRM fluorescence intensity. Fig. 8. Ca 2+ accumulation in mitochondria after MMPFs. Calcium Green-1 and TMRM images captured at the start and end of the illumination period. Bar is 10 lm. The plot shows the Calcium Green-1 fluorescence along a line (indicated in the image) crossing two mitochondrial filaments, before and after illumination. The hori- zontal width of mitochondrial profiles shown in the plot indicates that Ca 2+ influx is not accompanied by matrix swelling. Mitochondrial membrane potential fluctuations A. M. Falchi et al. 1656 FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS (Eugene, OR, USA). All other compounds were from Sigma (St Louis, MO, USA). Imaging Cells were observed with a 100·⁄1.0 water immersion objective, using a Zeiss Axioskop microscope (Oberkoken, Germany) with a HBO 50 W L)2 mercury lamp (Osram, Berlin, Germany) attenuated with a 0.3% transmittance neutral filter. Fluorescence filters were 528–552 ex, 580 sp., 590-LP em for TMRM; 460–500 ex, 505 sp., 510–560 em for DCDHF; 435–485 ex, 500 sp., 515–555 em for Calcium Green-1; 340–380 ex, 400 sp., 435–485 em for NAD(P)H. Images were acquired with a 12-bit cooled CCD camera (Sensicam; PCO Computer Optics, Kelheim, Germany) with a 1280 · 1024 pixel chip and 2 · 2 pixel binning. Opti- cal settings provided a nominal over-resolution of 0.1 lmÆ pixel )1 . TMRM images were acquired every 60 ms, in series of 230 images during 13.8 s of continuous illumination (16.67 frames per s). The series was truncated at the 230th image, as no MMPFs were observed after this time. TMRM images were preprocessed with a 3 · 3 average fil- ter to reduce random noise and unweighted time average (n ¼ 8) to reduce pixel replication noise. Calcium Green-1 images were acquired with an exposure of 1 s. Because of the relatively long period (12.5 s) of Ca 2+ oscillations induced by ATP, in these experiments the acquisition of images was prolonged to 1 min, at the rate of 1 frameÆs )1 . Induction of MMPFs Preliminary experiments showed that MMPFs depended on the intensity and duration of illumination and fluorescence intensity of mitochondria. Illumination conditions were eas- ily controlled, using the same optical settings in all experi- ments. On the other hand, the mitochondrial fluorescence was more difficult to control, because of conspicuous differ- ences in the amount of TMRM loaded by single cells [21]. Under a condition of continuous illumination required for fast image acquisition, MMPFs were optimally detected in mitochondria exhibiting a specific range of fluorescence intensity, represented by the gray value range 70–130. No MMPFs were found in mitochondria with a lower fluores- cence (gray value < 70). On the other hand, mitochondria with higher fluorescence (gray value > 130) displayed a very fast depolarization. In this case, the detection of MMPFs was hindered by a massive release of TMRM in the cytosol. These data were obtained under a condition of continuous illumination (20 ms exposure and 40 ms readout for each image). Mitochondrial depolarization and MMPFs were reduced when illumination was discontinuous, and completely abolished when the 20 ms illumination of a sin- gle image was followed by a dark period of 980 ms (one frameÆper second). The effect of probe bleaching under con- tinuous illumination was measured from the fluorescence decay of TMRM stains obtained by spraying microdroplets of 100 lm TMRM on a coverslip. TMRM stains were dried to avoid fluorescence recovery after photobleaching, and subsequently only those with a fluorescence intensity in the range of mitochondria (gray values 70–130) were selected for measurement. The effect of continuous and discontinu- ous illumination and bleaching are shown in Fig. 2A. Sampling Morphological criteria for the selection of mitochondria were the (a) perfect focus, (b) homogeneous thickness (no swelling or stretching), (c) separation from each other (no crossing), (d) sufficient extension to allow the measurement of three to five points along the filament, and (e) absence of movements. The occurrence of movements in the x–y plane and in the z-axis was carefully checked comparing the pixel positions and focus drift of mitochondria through the stack of images. However, owing to the relatively short duration of sessions and linear extension of mitochondrial filaments, the number of cases of exclusion was very small. Analysis of fluctuations D and R phases were identified by numerical differentiation obtained, for each time point, as the difference between the three preceding and the three following time points: FI 0 n ¼ðFI nÀ3 þ FI nÀ2 þ FI nÀ1 ÞÀðFI nþ1 þ FI nþ2 þ FI nþ3 Þ where FI is fluorescence intensity. This operation trans- formed D and R phases into peaks of different sign (posit- ive and negative, respectively), duration and intensity (Figs 2B,C and 3). The fluorescence change rate (ratio between fluorescence intensity change and duration) and frequency (number of events per mitochondrion per minute) were also calculated from the primary parameters. The overall noise (inclusive of random and stationary noise, due to current interference, instability of the arc lamp, occa- sional vibrations, etc.) was evaluated from the autofluores- cence of an inert plastic film, using the same optical settings (fluorescence filters, microscope magnification, iris opening, CCD gain, exposure, frame rate, etc.) and proce- dures (image processing, numerical methods) applied to cells. The fluorescence intensity of the plastic film was made physically equivalent to the average fluorescence intensity of TMRM by means of neutral density filters interposed between the lamp and the microscope. The max height (¼ 3) and max width (¼ 9) of derivative peaks of the plas- tic material were set as cut-off values to remove all noise fluctuations from TMRM data. However, it is possible that small MMPFs, not distinguishable from noise, may have been eliminated by the filtering method. The symmetry of derivative peaks was also taken into account to remove irregular fluctuations characterized by nonlinear slopes. Symmetry was calculated as the lowest of the reciprocal A. M. Falchi et al. Mitochondrial membrane potential fluctuations FEBS Journal 272 (2005) 1649–1659 ª 2005 FEBS 1657 ratios between the t1-p and p-t2 segments (Fig. 3). Sym- metry was 1 for perfectly centered peaks and less than 1, tending to zero, for increasingly irregular peaks, irrespective of the tail direction. The value of 0.333 was set as cut-off for symmetry. The relative frequency of rejections because of height, width and symmetry was 78%, 16% and 6%, respectively (Fig. 2D). Data were processed with specific routines developed for Microsoft Excel (Seattle, WA, USA) and Statistica (StatSoft, Tulsa, OK, USA). Owing to the considerable skewness of distributions, data were summar- ized by the median and the 25th)75th centile (interquartile) range. Differences were tested by analysis of variance followed by the Student–Newman–Keuls test for multiple comparisons. Acknowledgements We thank Professor Vincenzo Fiorentini (Physics Department, University of Cagliari) for helpful sugges- tions concerning numerical methods. The research was supported by grants from MIUR-FIRB (RBAU01C- CAJ_003), Istituto Zooprofilattico Sperimentale della Sardegna (IZS SA ⁄ 001 ⁄ 2001) and Regione Autonoma della Sardegna, Assessorato dell’Igiene e Sanita ` e dell’Assistenza Sociale. References 1 Loew ML, Tuft RA, Carrington W & Fay FS (1993) Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria. 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Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes J Cell Biol 142, 975–988 34 Ichas F & Mazat JP (1998) From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore Switching from low- to highconductance state Biochim Biophys Acta 1366, 33–50 35 Duchen MR (2000) Mitochondria and calcium: . Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation Angela. 2005) doi:10.1111/j.1742-4658.2005.04586.x Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester. data of single mitochondria, and some rep- resentative traces are shown in Figs 2B and 6. The exact identification of depolarization phases (D phases) and repolarization phases (R phases) of MMPFs