Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome‐ c release
2011; Springer Nature; Volume: 7; Issue: 1 Linguagem: Inglês
10.1038/msb.2011.2
ISSN1744-4292
AutoresHeinrich J. Huber, Heiko Düßmann, Seán M. Kilbride, Markus Rehm, Jochen H.M. Prehn,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle1 March 2011Open Access Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release Heinrich J Huber Heinrich J Huber Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Heiko Dussmann Heiko Dussmann Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Seán M Kilbride Seán M Kilbride Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Markus Rehm Markus Rehm Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Jochen H M Prehn Corresponding Author Jochen H M Prehn Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Heinrich J Huber Heinrich J Huber Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Heiko Dussmann Heiko Dussmann Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Seán M Kilbride Seán M Kilbride Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Markus Rehm Markus Rehm Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Jochen H M Prehn Corresponding Author Jochen H M Prehn Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Search for more papers by this author Author Information Heinrich J Huber1,‡, Heiko Dussmann1,‡, Seán M Kilbride1, Markus Rehm1 and Jochen H M Prehn 1 1Systems Biology Group, Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland ‡These authors contributed equally to this work *Corresponding author. Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland. Tel.: +353 1 402 2261; Fax: +353 1 402 2447; E-mail: [email protected] Molecular Systems Biology (2011)7:470https://doi.org/10.1038/msb.2011.2 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨm from −142 to −88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨm. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation. Synopsis How can cancer cells survive the consequences of cyt-c release? Huber et al provide a quantitative analysis of the protective role of enhanced glucose utilization in cancer cells and investigate the impact of cell-to-cell heterogeneity in mitochondrial bioenergetics. How can cells cope with a bioenergetic crisis? In particular, how can cancer cells survive the bioenergetic consequences of cyt-c release that are often induced by chemotherapeutic agents, and that lead to depolarisation of the mitochondrial membrane potential ΔΨm, result in loss of ionic homeostasis and induce cell death? Is there an inherent population heterogeneity that can lead to a non-synchronous response to above cell death stimuli, thereby aggravating treatment and contributing to clinical relapse? Do cancer cells have a competitive advantage to non-transformed cells in averting such a bioenergetic crisis after cyt-c release. We have investigated these questions in our study, which we regard as the first rigorous system study of single-cell bioenergetics subsequent to cyt-c release and one that bridges single-cell microscopy, in vitro analysis with ordinary differential equations (ODE) based modelling of bioenergetics pathways in the mitochondria and the cytosol. Several laboratories have so far investigated cyt-c release experimentally (Slee et al, 1999; Atlante et al, 2000; Goldstein et al, 2000; Luetjens et al, 2001; Plas et al, 2001; Waterhouse et al, 2001; Ricci et al, 2003; Colell et al, 2007; Dussmann et al, 2003a, 2003b) and isolated mitochondria (Chinopoulos and Adam-Vizi, 2009; Kushnareva et al, 2002; Kushnareva et al, 2001). However, the cause and mechanistic of several key findings remain elusive and need a system level understanding of post-cyt-c release bioenergetic and its potential cross-talk to apoptosis signalling. Ricci et al (2003) have identified that the cell death-inducing protease caspase-3, which get activated upon cyt-c release, can further impair mitochondrial function by cleaving and deactivating respiratory complexes I and II. We addressed the question of how such a mechanism could potentiate a bioenergetic crisis. To do so, we first assembled our ODE-based model by integrating approaches from metabolic modelling (Beard, 2005; Beard and Qian, 2007; Dash and Beard, 2008) and calibrated the model to literature data that describe bioenergetic state variables (mitochondrial membrane potential ΔΨm, mitochondrial transmembrane membrane ΔpH, respiration ratio between respiring and resting state mitochondria). By remodelling cyt-c release as observed experimentally and integrating it into our model as input, the single-cell model was able to correctly describe the kinetics of ΔΨm depolarisation and allowed its quantification. Moreover, it suggested that an additional complex I/II cleavage may further impair respiration and depolarise ΔΨm by approximately further 10%. It was further reported that ATP synthase reversal, a change of direction in the ATP-producing enzyme that leads to pumping of protons from the mitochondrial matrix into the intramembrane space, can stabilise ΔΨm. By a remnant ΔΨm polarisation, cycling of Na+, Ca2+, K+, Cl− ions and protons across the mitochondrial and the plasma membranes is preserved, and ionic homeostasis can be maintained (Nicholls and Budd, 2000; Dussmann et al, 2003a; Chinopoulos and Adam-Vizi, 2009; Garedew et al, 2010). Our model confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release, predicted that ATP synthase reversal consumed ATP and that glycolysis was required and sufficient to provide the necessary ATP to sustain this reversal. Reverting back to our single-cell HeLa system, we confirmed the presence of ATP synthase reversal. However, reversal was only present in 20% of cells, 65% of cells showed no detectable reaction and even 15% maintained ATP synthase in forward direction. To explain this cell-to-cell heterogeneity, we modelled that a cyt-c fraction remains accessible by respiratory complexes and for respiration subsequent to release, which we denoted as 'respiration-accessible cyt-c'. Our model suggested that small variations in such levels can sufficiently explain the experimentally detected population heterogeneity in the direction and amount of ATP synthase proton flux (Figure 6AB). Variations in respiration-accessible cyt-c may arise from incomplete mitochondrial release. Such incomplete release has been associated with failure of cristae remodelling in the absence of the BH3-only family member BID or the intramitochondrial protein OPA1 (Frezza et al, 2006; Scorrano et al, 2002). As the model identified glycolysis as necessary for sustaining ATP synthase reversal, we next investigated cells cultured in a medium that contained Na-pyruvate instead of glucose and which consequently were not able to perform glycolysis. We found that such cell populations had a significantly higher fraction of cells that maintained ATP synthase in forward mode consistent with our model predictions. The common influence of respiration-accessible cyt-c and the cell's ability to perform glycolysis is summarised in Figure 7A. Because glycolysis was able to influence ATP synthase proton pumping, which can affect ΔΨm levels, we investigating the effect of higher glucose in single cells. Our model predicted that an increase in glucose utilisation that generates higher cytosolic ATP levels is able to stabilise and repolarise ΔΨm and after release. This mechanism is independent from ATP synthase direction. For cells that have ATP synthase in reverse mode, elevated ATP leads to increased proton efflux from the matrix, cell populations that maintain ATP synthase in forward mode achieve a similar result through a reduction of proton influx at increased ATP. In both cases, the proton gradient along the inner membrane, and therefore ΔΨm, is increased as a consequence of ATP elevation. The mechanism is depicted in Figure 7B. We confirmed our model predictions that high glucose was able to stabilise (cells maintained in high-glucose media) and/or to repolarise (cells where glucose was added subsequent to release) ΔΨm (Figure 6). While a similar recovery was also present in MCF7 breast cancer cell lines, no significant effect of elevated glucose was found in non-transformed CRL-1807 cells. In conjunction with an impairment of caspase-dependent cell death observed in many human cancers, cancer cells may use this mechanism, and this mechanism may provide cancer cells with a competitive advantage to evade cell death induced by anticancer drugs or other stress conditions when compared with non-transformed cells. Introduction Cytochrome-c (cyt-c) is a key component of the mitochondrial respiratory chain, located in the intermembrane space (IMS) and responsible for transporting electrons from respiratory complex III to complex IV. By converting the redox potential established by glycolysis and the tricarboxylic acid cycle (TCA) into a proton gradient across the inner mitochondrial membrane, cyt-c is required to maintain polarisation of the mitochondrial transmembrane potential (ΔΨm). In turn, ΔΨm establishes a proton-motive force for the (FoF1-)ATP synthase to produce ATP (oxidative phosphorylation—OXPHOS; Mitchell, 1961; Nicholls and Ward, 2000), which preserves the cycling of Na+, Ca2+, K+, Cl− ions and protons across the mitochondrial and the plasma membranes to maintain ionic and osmotic homeostasis and to prevent necrotic cell death (Nicholls, 1977; Nicholls and Budd, 2000). To induce cancer cell death, chemotherapeutic agents often generate conditions such as genotoxic stress that lead to cyt-c release from the mitochondrial IMS into the cytosol, a process referred to as mitochondrial outer membrane permeabilisation (MOMP). This disrupts the mitochondrial respiratory chain and causes ΔΨm depolarisation, which in turn can result in a bioenergetic crisis characterised by ATP depletion, loss of ionic homeostasis, increased osmotic pressure and necrotic cell death (Jurgensmeier et al, 1998; Nicholls and Budd, 2000; Dussmann et al, 2003a). In addition, cyt-c release is also a direct transducer of apoptotic signals. Its presence in the cytosol enables the formation of the apoptosome, a heptameric complex of the cytosolic apoptotic protease-activating factor-1 and caspase-9 (Liu et al, 1996; Kluck et al, 1997), which activates effector caspases, in particular caspase-3 (Srinivasula et al, 1998; Slee et al, 1999). However, many cancer cells have developed strategies to survive both consequences of cyt-c release. Some cancer cells bypass caspase-dependent apoptosis through loss-of-function mutations or overexpression of caspase inhibitors. While the molecular mechanisms and systems aspects of impairment of caspase-dependent cell death are quite well understood (Deveraux and Reed, 1999; Rehm et al, 2006; Huber et al, 2007, 2010), the means by which cancer cells can survive, despite cyt-c release-induced bioenergetic crisis, remain more elusive. The complexity of bioenergetic pathways can barely be captured by traditional studies that focus on a single metabolite or protein at a time. Moreover, interdependencies between mitochondrial and cellular bioenergetics and caspase-dependent cell death have been identified (Matsuyama et al, 2000; Ricci et al, 2003), posing the need for their coinvestigation in a holistic approach. In this report, we present the first integrated systems biology study of mitochondrial bioenergetics and apoptosis and bridge the gap between metabolic modelling and a single-cell experimental analysis. We developed a computational model that integrates existing knowledge from metabolic engineering (Beard 2005; Korzeniewski and Brown, 1998) with our recently established, ordinary differential equations (ODEs) based model of the mitochondrial apoptosis pathway (Rehm et al, 2006; Huber et al, 2007). Our approach and findings are summarised by the chart shown in Figure 1. Challenging the model with single-cell experiments, we remodelled the kinetics of mitochondrial depolarisation after cyt-c release in the presence or absence of caspase activation. Mathematical modelling and experimental validation identified glycolysis and variations in the amount of cyt-c that remains accessible for respiration at the mitochondria to be the key factors in determining the ability of cancer cells to prevent a bioenergetic crisis post-cyt-c release. Figure 1.Organisation scheme of the combined single-cell microscopy and in silico analysis. The scheme shows a workflow diagram of the procedure that was pursued as outlined in the main text. Results of in silico modelling are shown in blue boxes. Red boxes indicate experimental validations as performed by single-cell microscopy or by data obtained from the literature on isolated mitochondria. Green boxes depict research findings that were identified within the text and which resulted from challenging the in silico predictions with experimental data and vice versa. Download figure Download PowerPoint Results Model calibration to in vitro data for ATP-producing and non-ATP-producing mitochondria We devised our model with gradually increasing complexity and thus started with the widely studied experimental system of isolated mitochondria. We assembled the network of electrochemical reactions consisting of mitochondrial respiration, ATP production and ion transport, and used a fixed NADH/NAD disequilibrium as model input (Figure 2, and Materials and methods section). By Monte-Carlo screening, we calibrated the model into three scenarios that have been experimentally well described. These were (i) ATP producing ('state-3') mitochondria in buffered medium of an ATP/ADP ratio of 3:1 (75% ATP, 25% ADP), (ii) non-ATP producing, resting-state ('state-4') mitochondria (100% ATP, no ADP) and (iii) a situation where we considered mitochondria to change from state-3 to state-4 (Supplementary Figure 1A). Further details on the model construction and its calibration can be found in Supplementary Text I. Figure 2.Computational model of mitochondrial bioenergetics during cyt-c release and apoptosis. Interactions and transport processes between the mitochondrial matrix ('Matrix'), mitochondrial intermembrane space ('IMS') and the cytosol ('Cytosol'). Roman numbers indicate single-cell processes that influence mitochondrial bioenergetics: (I) model of mitochondrial apoptosis (according to Rehm et al, 2006) that activates caspase-3 upon cyt-c release; (II) cytosolic ATP production and consumption; (III) active caspase-3 cleaving complex I/II; (IV) cyt-c release. Metabolite and ion fluxes of mitochondrial bioenergetics are given by Arabic numbers: (1) input function (NADH/NAD disequilibrium=45.8:1); (2–4) respiration complexes I/II (considered together), III and IV; (5) ATP synthase; (6) adenosine nucleotide transferase (ANT); (7) mitochondrial inner membrane proton leaks; (8) passive transport of adenosine phosphates and anorganic phosphate trough the outer mitochondrial membrane; (9) phosphate–proton cotransport and (10) proton–potassium antiport. Download figure Download PowerPoint Our best fit led to a respiratory control ratio (state-3/state-4 respiration ratio) of 6.5:1, which is in agreement with reported values of 5–10 (Nicholls, 1974a, 1974b; Nicholls and Ward, 2000). ΔΨm values were calculated to be −157 mV for state-4 and −137 mV for state-3, consistent with literature values of ΔΨm from −140 to −150 mV (Nicholls and Budd, 2000) and with a reported 10–15% increase in ΔΨm polarisation when mitochondria switch from state-3 to state-4 on ATP replenishment (Nicholls, 1974). Transmembrane ΔpH between the mitochondrial matrix and the mitochondrial IMS was calculated as 0.41 for state-3 and 0.32 for state-4 (Supplementary Figure 1B). In the case where we modelled state-3 to state-4 transitions, we achieved ATP replenishment within 2 min in accordance with the well-established rapid kinetics of ATP production, where a 90% equilibrium was reached in about 1 min (Pfaff et al, 1969; Supplementary Figure 1C for ATP/ADP ratio 1D for respiration flux and ΔΨm, 1E for ATP synthase proton flux and activity of constitutive proton leaks over time). A detailed sensitivity analysis of the influence of model parameters (Supplementary Table VII) on calibration results is given in Supplementary Figure 2. A parameter cluster analysis (Gutenkunst et al, 2007) allowed to characterise the model mechanistic and identified four principal components that explain 94% of the model parameter variations (Supplementary Figure 3). Further details on the model mechanistic and sensitivity analysis can be found in Supplementary Text II. Cyt-c release impairs respiratory flux and leads to mitochondrial depolarisation Mitochondrial cyt-c release following MOMP is considered as a point of no return in the mitochondrial apoptosis pathway. Loss of cyt-c disrupts the mitochondrial electron transport chain between complex III and IV, depolarises ΔΨm and impairs ATP production. Once in the cytosol, cyt-c leads to activation of the apoptosome-dependent cell death pathway and caspase-3 activation (Green and Kroemer, 2004). However, evidence has been presented that mitochondria and cellular bioenergetics can recover after the release of cyt-c, particularly when effector caspase activation is impaired (Waterhouse et al, 2001; Dussmann et al, 2003a; Colell et al, 2007). These studies suggested that cells can survive a MOMP-dependent energy crisis subsequent to cyt-c release. We used the model to investigate the effect of cyt-c release on respiration and ΔΨm in the presence or absence of active caspases. Cyt-c release was modelled to start at time t=10 min after assuming cells in equilibrium. The kinetic parameters of cyt-c release were extracted from previous single-cell measurements (see Materials and methods section; Goldstein et al, 2000; Luetjens et al, 2001; Waterhouse et al, 2001; Dussmann et al, 2003a, Figure 3A, dotted green line). We assumed re-equilibration of cyt-c between the IMS and the cytosol, and therefore concluded that a final cyt-c fraction of 0.1% remained accessible for respiration (see Method section). We have hitherto denoted this cyt-c fraction as 'respiration-accessible cyt-c'. Figure 3.Live cell imaging and computational modelling of cyt-c release and mitochondrial depolarisation in HeLa cells expressing cyt-c–GFP. (A) Remodelling of experimentally observed cyt-c release at time point t=10 min (Goldstein et al, 2000; Waterhouse et al, 2001; Dussmann et al, 2003a) with a half-time of 1.5 min and a remnant value of 0.1% (green dotted line), as described in the text, leads to active caspase-3 (black solid line) and deactivation of complex I/II (according to the model of Rehm et al, 2006) within 15–20 min after onset of cyt-c release (blue dashed line). (B) Calculated depolarisation of mitochondrial membrane potential as a consequence of cyt-c release as given in (A) with or without caspase-3-induced complex I/II cleavage (dashed red and solid blue line). (C, D) Representative single-cell traces of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and exposed to 3 μM staurosporine (STS). (C) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM (both normalised to initial values) of the two labelled cells in (E). (E) Fluorescence images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D, F) As in (C, E), but in the presence of 100 μM of the caspase inhibitor zVAD-fmk administrated 1 h prior to exposure to 3 μM STS. Source data is available for this figure at www.nature.com/msb. Source data for Figure 3C [msb20112-sup-0002-SourceData-S2.xls] Source data for Figure 3D [msb20112-sup-0003-SourceData-S3.xls] Download figure Download PowerPoint We further modelled cyt-c to induce apoptosome formation and to activate caspase-3 after release. Therefore, we included our previously established model of mitochondrial apoptosis (Rehm et al, 2006) as a code subroutine and calculated the level of active caspase-3 over time. The model predicted caspase-3 activation and deactivation of complex I/II as previously reported (Ricci et al, 2003; Figure 3A, solid back and blue dashed lines, respectively) within 10–15 min of cyt-c release. As another consequence of cyt-c release, respiratory flux was decreased after onset of cyt-c release to 5% within 10–15 min, while a further decrease to 1% was observed when caspase-3 was active (see Supplementary Figure 4A for a mechanistic scheme and 4B for results). Depolarisation to a remnant ΔΨm of −88 or −76 mV was evident in the absence or presence of active caspase-3, respectively (Figure 3B). ΔΨm depolarisation after cyt-c release and further depolarisation by active caspase-3 were robust over a wide range of model parameter variations (see Supplementary Figure 5 and Supplementary Text II for a sensitivity analysis). To validate the kinetics of ΔΨm depolarisation and the effect of active caspase-3 in cellulo, we performed single-cell imaging experiments using HeLa cells expressing cyt-c–green fluorescent protein (GFP), which we incubated with 30 nM tetramethyl rhodamine methyl-ester (TMRM). Cyt-c release was induced with 3 μM of the broad-spectrum kinase-inhibitor staurosporine (STS). Indeed, these experiments suggested a greater degree of mitochondrial depolarisation in the absence of a broad-spectrum caspase-inhibitor Z-Val-Ala-Asp(O-methyl)-fluoromethylketone (zVAD-fmk; Figure 3C and E) than in its presence (Figure 3D and F). This indicates a role of caspase-3 in ΔΨm depolarisation after cyt-c release, although the influence of changes in plasma membrane potential on TMRM redistribution upon caspase activation cannot be excluded (Dussmann et al, 2003a). In summary, the single-cell model mechanistically described and quantified ΔΨm depolarisation as measured in living cells subsequent to cyt-c release. Moreover, it suggested that an additional complex I/II cleavage may further impair respiration and ΔΨm. Characterisation of ATP synthase reversal as a stabiliser of ΔΨm Maintenance of ΔΨm subsequent to cyt-c release is essential for ionic homeostasis within the cell and thus may prevent the onset of necrotic cell death (Nicholls, 1977; Nicholls and Budd, 2000). Reversal of ATP synthase has been proposed to stabilise ΔΨm by pumping protons from the matrix into the IMS, while consuming instead of generating ATP (Goldstein et al, 2000; Chinopoulos and Adam-Vizi, 2009). However, it has also been reported that ΔΨm can stabilise in the absence of ATP synthase reversal. We therefore aimed to investigate whether and under what conditions ATP synthase reversal was present and if so what the implications on cytosolic ATP and ΔΨm were. We first confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release when respiration-accessible cyt-c decreased to 0.1% in our model (Figure 4A, blue solid line). This was slightly more pronounced when caspase-3 deactivation of complex I/II was also taken into consideration (Figure 4A, red dashed line). Moreover, our model confirmed that ATP synthase reversal consumed ATP (Supplementary Figure 6A), that glycolysis prevented ATP depletion during this reversal (Supplementary Figure 6B) and that ATP synthase reversal was robust when model parameters (Supplementary Table VII) were increased or decreased over a fivefold parameter range (Supplementary Figure 7A and Supplementary Text II). Figure 4.Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb. Source data for Figure 4D [msb20112-sup-0004-SourceData-S4.xls] Download figure Download PowerPoint We then examined whether ATP synthase reversal stabilised ΔΨm, as was previously suggested (Rego et al, 2001; Dussmann et al, 2003a). We therefore set ATP synthase activity to zero in our single-cell model at 30 min after onset of cyt-c release. Inhibition of the reversed ATP synthase (as in Figure 4A) predicted the occurrence of a further mitochondrial depolarisation from −88 to −65 mV (−76 to −26 mV when considering complex I/II inhibition by caspase-3, Figure 4B). Owing to the lower ΔΨm and a resulting lower electrostatic barrier for proton pumping through respiratory complexes, a slight regain of respiratory flux (Supplementary Figure 6C) was also detected. To experimentally validate the presence of reversed ATP synthase, we added 5 μM of the ATP synthase inhibitor oligomycin to HeLa cyt-c–GFP cells subsequent to cyt-c release. Indeed, oligomycin induced a further ΔΨm depolarisation within 30 min (Figure 4C and D) indicating reverse ATP synthase activity, albeit in only 20% of a total of 237 cells (n=6 experiments). Overall, 65% of cells showed no detectable reaction. Strikingly, about 15% even showed a partial repolarisation immediately after administration of oligomycin (Figure 4E) suggesting that ATP synthase was maintained in forward mode in these cells. In all cells, the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) could fully depolarise ΔΨm to negligible levels, demonstrating that mitochondria maintained a remnant ΔΨm. In conclusion, ATP synthase reversal was found in some cells to stabilise ΔΨm by consuming ATP after cyt-c release, yet an unexpectedly wide cell-to-cell heterogeneity was observed with regard to the direction of ATP synthase activity. Respiration-accessible cyt-c subsequent to MOMP influences activity and direct
Referência(s)