Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent
2019; Springer Nature; Volume: 38; Issue: 22 Linguagem: Inglês
10.15252/embj.2018101056
ISSN1460-2075
AutoresDane M. Wolf, Mayuko Segawa, Arun Kumar Kondadi, Ruchika Anand, Sean T. Bailey, Andreas S. Reichert, Alexander M. van der Bliek, David B. Shackelford, Marc Liesa, Orian S. Shirihai,
Tópico(s)Metabolism and Genetic Disorders
ResumoArticle14 October 2019Open Access Source DataTransparent process Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent Dane M Wolf Dane M Wolf Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Graduate Program in Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Mayuko Segawa Mayuko Segawa Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Arun Kumar Kondadi Arun Kumar Kondadi Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Ruchika Anand Ruchika Anand Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Sean T Bailey Sean T Bailey orcid.org/0000-0001-8051-6960 Department of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Andreas S Reichert Andreas S Reichert Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Alexander M van der Bliek Alexander M van der Bliek orcid.org/0000-0002-6211-5765 Molecular Biology Institute at UCLA, Los Angeles, CA, USA Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author David B Shackelford David B Shackelford Department of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Marc Liesa Corresponding Author Marc Liesa [email protected] orcid.org/0000-0002-5909-8570 Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Molecular Biology Institute at UCLA, Los Angeles, CA, USA Search for more papers by this author Orian S Shirihai Corresponding Author Orian S Shirihai [email protected] orcid.org/0000-0001-8466-3431 Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Graduate Program in Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Dane M Wolf Dane M Wolf Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Graduate Program in Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Mayuko Segawa Mayuko Segawa Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Arun Kumar Kondadi Arun Kumar Kondadi Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Ruchika Anand Ruchika Anand Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Sean T Bailey Sean T Bailey orcid.org/0000-0001-8051-6960 Department of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Andreas S Reichert Andreas S Reichert Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany Search for more papers by this author Alexander M van der Bliek Alexander M van der Bliek orcid.org/0000-0002-6211-5765 Molecular Biology Institute at UCLA, Los Angeles, CA, USA Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author David B Shackelford David B Shackelford Department of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Search for more papers by this author Marc Liesa Corresponding Author Marc Liesa [email protected] orcid.org/0000-0002-5909-8570 Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Molecular Biology Institute at UCLA, Los Angeles, CA, USA Search for more papers by this author Orian S Shirihai Corresponding Author Orian S Shirihai [email protected] orcid.org/0000-0001-8466-3431 Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Graduate Program in Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Author Information Dane M Wolf1,2,†, Mayuko Segawa1,†, Arun Kumar Kondadi3,‡, Ruchika Anand3,‡, Sean T Bailey4,5,6, Andreas S Reichert3, Alexander M Bliek7,8, David B Shackelford4,5, Marc Liesa *,1,7,§ and Orian S Shirihai *,1,2,§ 1Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 2Graduate Program in Nutrition and Metabolism, Graduate Medical Sciences, Boston University School of Medicine, Boston, MA, USA 3Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 4Department of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 5Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, CA, USA 6Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 7Molecular Biology Institute at UCLA, Los Angeles, CA, USA 8Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA †These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as second authors §These authors contributed equally to this work as corresponding authors *Corresponding author. Tel: +1-310-206-7319; E-mail: [email protected] *Corresponding author. Tel: +1-617-230-8570; E-mail: [email protected] The EMBO Journal (2019)38:e101056https://doi.org/10.15252/embj.2018101056 See also: M Schlame (November 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mitochondrial membrane potential (ΔΨm) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm. However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm, corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm. Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest. Synopsis Mitochondrial membrane potential (ΔΨm) is the main driving force for ATP synthesis at the folds of the inner mitochondrial membrane, the cristae. Measurement of ΔΨm in individual cristae reveals that crista junctions provide electrical insulation and sustain polarization of individual mitochondrial cristae within a single mitochondrion even when neighbouring cristae are damaged. Cristae have higher ΔΨ compared to their adjoining inner mitochondrial membranes. Cristae are electrically insulated, allowing individual cristae within any given mitochondrion to have different membrane potentials. Cristae can remain polarized despite depolarization of neighbouring ones. Disruption of crista junctions impairs the electrical insulation of cristae, equilibrating their ΔΨ with those of inner mitochondrial membranes. Introduction Mitochondria utilize nutrients and molecular oxygen to generate a membrane potential (ΔΨm) across the inner mitochondrial membrane (IMM). The energy available for ATP synthesis is directly derived from ΔΨm (Mitchell, 1961; Mitchell & Moyle, 1969); therefore, depolarization directly translates to decreased energy availability for ATP synthesis. Classical studies suggested that the ΔΨm was homogeneous along the IMM. Data supporting the equipotential model are (i) mitochondria labeled with ΔΨm-dependent dyes show a homogeneous signal along a single mitochondrion visualized under a low resolution microscope, indicating that the ΔΨm is likewise homogeneous all along the organelle (Amchenkova et al, 1988; Skulachev, 2001); and (ii) an elongated mitochondrion stained with a ΔΨm-dependent dye appears to instantaneously lose its ΔΨm following laser-induced damage to a small (≤ 0.5 μm2) region, suggesting that a mitochondrial filament is analogous to a power cable, where, if one part is compromised, the voltage will simultaneously collapse across its entire length (Amchenkova et al, 1988; Skulachev, 2001; Glancy et al, 2015). These conclusions were drawn after imaging mitochondria with ΔΨm-dependent dyes performed with light microscopes lacking sufficient spatial resolution to visualize the ultrastructure of the IMM. Furthermore, previous studies lacked the temporal resolution to determine whether laser-induced depolarization leads to an instantaneous collapse of ΔΨm across the whole organelle. The IMM consists of subcompartments called cristae and inner boundary membrane (IBM) (Palade, 1953). Cristae are invaginations protruding into the mitochondrial matrix, whereas the IBM runs parallel to the outer mitochondrial membrane (OMM). Cristae and IBM are connected via narrow tubular or slit-like structures, known as crista junctions (CJs). In recent years, studies show that components of the electron transport chain (ETC) are confined to the lateral surfaces of the cristae rather than equally distributed along the IMM (Vogel et al, 2006; Wilkens et al, 2013). Moreover, dimers of F1F0 ATP Synthase assemble in rows along the edges of the cristae (Dudkina et al, 2005; Strauss et al, 2008; Davies et al, 2011). The CJs can be kept in a closed state by oligomers of the inner-membrane dynamin-like GTPase, OPA1 (Frezza et al, 2006; Pham et al, 2016), as well as components of the mitochondrial contact site and cristae organizing system (MICOS complex) (John et al, 2005; Rabl et al, 2009; Barrera et al, 2016; Glytsou et al, 2016). These findings provide a conceptual framework, where protons pumped by the ETC across the cristae membrane appear first in the cristae lumen (Busch et al, 2013; Pham et al, 2016). However, unless the ΔΨ of the crista membrane is kept more negative compared to its neighboring IBM, protons would not remain in the cristae. This consideration implies that differences in ΔΨm would exist between the cristae membrane and the IBM. To establish whether the distribution and structural properties of OXPHOS complexes are functionally significant, it would be critical to directly visualize and quantify the ΔΨm in relation to the IMM in living cells. If the ΔΨm is uniform from one end of a mitochondrion to another, the ΔΨm would be equal at any point along the IMM, supporting the equipotential model. If, however, the ΔΨm stems from cristae functioning as independent and heterogeneous compartments, the ΔΨm would vary substantially along the IMM—between cristae and IBM, as well as between different cristae. Testing such hypotheses, nonetheless, has remained virtually unfeasible, because the only way to resolve the IMM has been with the electron microscope, which requires freezing or fixation of mitochondria and therefore precludes any direct measurement of structure and ΔΨm. To overcome this limitation, we developed a novel approach for imaging the IMM at high spatiotemporal resolution in living cells, using the LSM880 with Airyscan as well as STED microscopy. Staining active mitochondria with various dyes, we verified that we can resolve cristae from IBM. We then used various ΔΨm-dependent dyes to explore how the intricate architecture of the IMM relates to the most basic mitochondrial function—the ΔΨm generated by the electrochemical gradient of protons. Results Development of an Airyscan-based approach to resolve cristae and IBM in living cells Previous studies show that components of OXPHOS are unevenly distributed between the cristae and IBM (Vogel et al, 2006; Wilkens et al, 2013), suggesting the possibility of ΔΨm heterogeneity along the IMM within a single mitochondrion. To develop an approach for the imaging of ΔΨm associated with cristae and IBM, we first sought to determine whether we could resolve the compartmentalization of the IMM in living cells. To address this question, we incubated various cell types with 10-N-nonyl acridine orange (NAO), a fluorescent probe that preferentially binds cardiolipin but also shows some affinity for other phospholipids found in mitochondria, such as phosphatidylethanolamine (PE) and phosphatidylinositol (PI) (Leung et al, 2014). Imaging mitochondria from living HeLa, L6, and H1975 cells with the LSM880 equipped with Airyscan technology, we resolved intramitochondrial structures, typically perpendicular to the long axis of the mitochondrion, resembling cristae, as observed in electron micrographs (Fig 1A). Accordingly, the high resolution of the Airyscan-based microscopy allowed separation of cristae structures, IBM, as well as dimmer regions, appearing to be matrix (Fig 1B). To verify that they were matrix, we used matrix-targeted DsRed to label the matrix in H1975 cells and stained their IMM with NAO (Fig 1C). Airyscan imaging confirmed that the dimmer regions of NAO fluorescence within the mitochondria showed the strongest matrix-DsRed signal and vice versa (arrowheads). To confirm that we correctly identified the cristae structures in cells stained with NAO, we tested whether the pattern of NAO labeling was changed in cells with disrupted cristae structure. As a model, we used HeLa cells with Crispr/Cas9-mediated KO of Mic13 (Fig EV1A), which destabilizes CJs and disrupts cristae structure (Fig 1D) (Anand et al, 2016; Guarani et al, 2015). Compared to control HeLa (Fig 1A and B), Mic13-KO mitochondria showed a substantial decrease in the number of perpendicular structures, supporting their identification as cristae. As a second model of cristae perturbation, we examined H1975 cells with stable KD of PTPMT1 through lentiviral transduction encoding shRNA (Fig EV1B). PTPMT1 is a mitochondrial phosphatase, essential for biosynthesis of phosphatidylglycerol, a precursor of cardiolipin. Deletion of PTPMT1 has been shown to result in severe derangement of the IMM (Zhang et al, 2011). Our Airyscan imaging of sh-Scramble (sh-Scr) control mitochondria shows structures closely resembling normal cristae (Fig 1E), whereas sh-PTPMT1 mitochondria display a variety of deformed structures (Fig 1F) analogous to cristae perturbations that were previously observed in electron micrographs of PTPMT1-deficient models (Zhang et al, 2011). Overall, these data demonstrate that Airyscan technology can resolve mitochondrial ultrastructure in living cells. We subsequently used this approach to measure ΔΨm at the different compartments along the IMM and determine the level of heterogeneity in ΔΨm within the individual mitochondrion. Figure 1. High-resolution fluorescence imaging using Airyscan resolves the inner mitochondrial membrane (IMM) structure in live cellsHigh-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. A. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type. B. Mitochondrion cropped from HeLa cell shown in (A) (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm. C. H1975 cells transduced with matrix-targeted DsRed and stained with NAO. Matrix-targeted DsRed differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment. D. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel (A), labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments. E, F. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. (E) A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. (F) A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Validation of KO and KD approaches of Cristae structure and CJ modulators Representative image of Western blot, showing deletion of MIC13 in HeLa cells. N = 3 independent experiments. Representative image of Western blot, showing deletion of PTPMT1 in H1975 cells. N = 3 independent experiments. Representative image of Western blot, showing deletion of MIC60 and MIC10 in Hap1 cells. N = 3 independent experiments. Representative image of Western blot, showing Opa1 KO and Opa1&Drp1 DKO in MEFs. N = 3 independent experiments. Source data are available online for this figure. Download figure Download PowerPoint ΔΨm-dependent dyes colocalize most strongly with cristae The power-cable model of the ΔΨm assumes an electrical continuity along the IMM without any electrical resistance (Skulachev, 2001). However, the possibility of heterogeneity in ΔΨm along the IMM could not be investigated until now. To image ΔΨm along the IMM, we stained HeLa cells with NAO and TMRE (Farkas et al, 1989; Loew et al, 1993). Remarkably, TMRE appeared to align with the IMM in a non-homogeneous manner, where the most-intense TMRE signal colocalized with NAO at cristae (Fig 2A). To substantiate this observation, we looked at L6 (rat myoblast) cells, which presented the same heterogeneous pattern as HeLa cells (Fig 2B). Figure 2. The ΔΨm-sensitive dye, TMRE, partitions to cristae stained with NAOHigh-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that, mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments. Download figure Download PowerPoint When excited by a 488-nm laser, NAO has an emission spectrum that is limited to green wavelengths. However, in these experiments, we needed to excite NAO with the 488-nm laser while simultaneously exciting TMRE with the 561-nm laser, resulting in NAO being exposed to both 488- and 561-nm lasers. The observed alignment of TMRE signal with the membrane staining by NAO raised the possibility that exciting NAO with the 561-nm laser could result in red light emission and thus be wrongly detected as TMRE. To address this possibility, we imaged cells stained with NAO alone and excited simultaneously with 488- (NAOEX) and 561-nm (TMREEX) lasers (Fig 2C, top row). We found that emission of NAO after excitation with the 561-nm laser (TMREEX) was undetectable. After adding TMRE to the cells, initially stained with NAO alone, and then exciting with the 561-nm laser using the same power, we observed the appearance of strong signal in the red channel, with most-intense pixels colocalizing with NAO at cristae (Fig 2C, bottom row). To further validate our findings with TMRE and NAO, we used two additional dyes, MitoTracker Green (MTG) and Rhodamine123 (Rho123). MTG covalently binds to various proteins embedded in the cristae membrane and, as such, is considered a ΔΨm-independent dye, although its initial sequestration in mitochondria depends on ΔΨm (Presley et al, 2003). Rho123 is a ΔΨm probe, which partitions to mitochondria in a transient way, indicating changes to ΔΨm (Ward et al, 2000; Duchen, 2004). We found that MTG colocalized with TMRE, showing a similar heterogeneous pattern (Fig EV2A). Then, we examined the partitioning of Rho123 and found that it shows the most-intense signal associated with cristae (Fig EV2B). Click here to expand this figure. Figure EV2. Membrane potential dyes, TMRE and Rho123, are concentrated at the cristae membranes in a heterogeneous pattern Mitochondria in HeLa cells co-stained with MTG and TMRE. Zoomed-in region from dashed white box highlights colocalization of dyes, showing that signal intensities vary together across the long axis (green and red lines); arrowheads indicate cristae membranes. Scale bar = 500 nm. N = 3 independent experiments. Mitochondria in HeLa cell stained with ΔΨm-dependent dye, Rho123; zoomed-in mitochondrion cropped from dashed line highlights more intense signal at cristae (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Download figure Download PowerPoint To further verify that the staining patterns of TMRE depend on ΔΨm, we used a previously described method to influence ΔΨm: Continuous exposure of TMRE-stained mitochondria to the 488-nm laser results in robust and rapid depolarization and repolarization, a phenomenon known as flickering (Duchen et al, 1998). We reasoned that the smaller portion of TMRE bound to the membrane in a ΔΨm-independent manner would remain during the flickering event and reveal the level of noise. Moreover, if the differences in TMRE fluorescence intensity (FI) between cristae and IBM depend on ΔΨm, we would expect that the differences between the brightest and dimmest pixels would markedly decrease during depolarization. Conversely, following repolarization, we would expect these differences in pixel intensities to return. Figure 3A shows an example of a mitochondrion from a HeLa cell stained with MTG and TMRE (arrowheads), where we initially observed the heterogeneous patterns of TMRE; however, at ~ 9 s, the mitochondrion depolarized, and the heterogeneous staining pattern of TMRE was lost. Notably, at ~ 16 s, this mitochondrion repolarized and exhibited nearly the same TMRE heterogeneity as before the depolarization. Quantification of the changes in ΔΨm during the flickering phenomenon demonstrates that ~ 85% of TMRE signal was lost during depolarization (Fig 3B). Moreover, the differences between the brightest and dimmest areas (cristae and matrix, respectively) are attenuated during transient depolarization but are reestablished following restoration of ΔΨm. These data support that the heterogeneous staining patterns of the TMRE are due to differences in ΔΨm. Figure 3. Validation that TMRE partitioning to cristae is ΔΨm-dependent Time-lapse Airyscan imaging of ΔΨm in living HeLa cell, co-stained with MTG (ΔΨm-insensitive after loading) and TMRE (ΔΨm sensitive). Arrowhead points to a flickering event where a mitochondrion depolarizes (˜ 9 s) and repolarizes (˜ 16 s), showing that heterogeneous signal from TMRE (but not MTG) disappears and reappears. Scale bar = 500 nm. N = 4 independent experiments. Quantification of (A). Plot shows average TMRE fluorescence intensity (FI) of cristae (dark red line) vs. matrix (light red line) during the time series. The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm-sensitive component of the TMRE signal. The remaining TMRE FI during depolarization can be considered as the ΔΨm-insensitive portion of TMRE signal. Note that, the remaining TMRE FI after depolarization at the cristae and matrix is approximately identical, indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm. N = 4 independent experiments. Data information: Data were analyzed with 2-tailed Student's t-tests, and P values < 0.05 were considered statistically significant. Specific P values are indicated in the figure. Error bars indicate SEM. Download figure Download PowerPoint Quantification of ΔΨm differences between cristae and IBM The Nernst equation can be used to quantify ΔΨm by acquiring the FI of ΔΨm-sensitive probes (e.g., TMRE). The FIs of the probes at different subcellular compartments can be used to extrapolate the differences in concentrations of the probe, which are needed to calculate the difference in ΔΨm between compartments (Ehrenberg et al, 1988; Farkas et al, 1989; Loew et al, 1993; Wikstrom et al, 2007; Twig et al, 2008). We used the average TMRE FI of the mitochondria as a reference point to calculate the ΔΨm of the different compartments, an approach similar to that employed in multi-electrode ECG. We found the voltage at cristae to be significantly higher than at IBM (Fig 4A and C). These data indicate that the hetero-potential along the IMM consists of at least two basic segments—the cristae and the IBM. Representing pixel intensities in pseudo-color as a LookUp Table (LUT), where white and blue correspond to the highest and lowest ΔΨm, respectively, it is apparent that the voltage associated with cristae (arrowheads) is generally higher than IBM, emphasizing the electrochemical discontinuity between these contiguous regions of the IMM (Fig 4B). Figure 4. Cristae and IBM have different ΔΨm A. Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. B. LUT of mitochondrion in HeLa cell shown in (A), color coding of TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that, most-intense pixels (white) only associate with cristae. C. Quantification of ΔΨm (mV) at cristae and IBM relative t
Referência(s)