Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent

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.


Introduction
Mitochondria utilize nutrients and molecular oxygen to generate a membrane potential (DΨ m ) across the inner mitochondrial membrane (IMM). The energy available for ATP synthesis is directly derived from DΨ m (Mitchell, 1961;Mitchell & Moyle, 1969); therefore, depolarization directly translates to decreased energy availability for ATP synthesis.
Classical studies suggested that the DΨ m was homogeneous along the IMM. Data supporting the equipotential model are (i) mitochondria labeled with DΨ m -dependent dyes show a homogeneous signal along a single mitochondrion visualized under a low resolution microscope, indicating that the DΨ m is likewise homogeneous all along the organelle (Amchenkova et al, 1988;Skulachev, 2001); and (ii) an elongated mitochondrion stained with a DΨ mdependent dye appears to instantaneously lose its DΨ m following laser-induced damage to a small (≤ 0.5 lm 2 ) 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 DΨ m -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 DΨ m 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 F 1 F 0 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 Pham et al, 2016). However, unless the DΨ 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 DΨ m 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 DΨ m in relation to the IMM in living cells. If the DΨ m is uniform from one end of a mitochondrion to another, the DΨ m would be equal at any point along the IMM, supporting the equipotential model. If, however, the DΨ m stems from cristae functioning as independent and heterogeneous compartments, the DΨ m 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 DΨ m .
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 DΨ m -dependent dyes to explore how the intricate architecture of the IMM relates to the most basic mitochondrial function-the DΨ m generated by the electrochemical gradient of protons.

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 DΨ m heterogeneity along the IMM within a single mitochondrion. To develop an approach for the imaging of DΨ m 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 matrixtargeted 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 ▸ Figure 1. High-resolution fluorescence imaging using Airyscan resolves the inner mitochondrial membrane (IMM) structure in live cells.
High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope.  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 DΨ m at the different compartments along the IMM and determine the level of heterogeneity in DΨ m within the individual mitochondrion.
DΨ m -dependent dyes colocalize most strongly with cristae The power-cable model of the DΨ m assumes an electrical continuity along the IMM without any electrical resistance (Skulachev, 2001). However, the possibility of heterogeneity in DΨ m along the IMM could not be investigated until now. To image DΨ m 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). 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-(NAO EX ) and 561-nm (TMRE EX ) lasers ( Fig 2C, top row). We found that emission of NAO after excitation with the 561-nm laser (TMRE EX ) 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 DΨ m -independent dye, although its initial sequestration in mitochondria depends on DΨ m (Presley et al, 2003). Rho123 is a DΨ m probe, which partitions to mitochondria in a transient way, indicating changes to DΨ m (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).
To further verify that the staining patterns of TMRE depend on DΨ m , we used a previously described method to influence DΨ m : 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 DΨ m -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 DΨ m , 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 DΨ m 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 DΨ m . These data support that the heterogeneous staining patterns of the TMRE are due to differences in DΨ m .

Quantification of DΨ m differences between cristae and IBM
The Nernst equation can be used to quantify DΨ m by acquiring the FI of DΨ m -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 DΨ m 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 DΨ m 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 heteropotential 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 DΨ m , respectively, it is apparent that the voltage associated with cristae (arrowheads) is generally higher than IBM, ◀ Figure 2. The DΨ m -sensitive dye, TMRE, partitions to cristae stained with NAO.
High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope.
A 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. B 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. C Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488and 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.
ª 2019 The Authors The EMBO Journal 38: e101056 | 2019 emphasizing the electrochemical discontinuity between these contiguous regions of the IMM (Fig 4B). We next explored whether the DΨ m of primary cells would follow the same pattern. We stained mitochondria in primary hepatocytes with TMRE and found DΨ m heterogeneity similar to that in HeLa cells, indicating that discrete electrochemical domains also exist along the IMM of differentiated cells with strong mitochondrial oxidative function (Fig 4D-F).
To further confirm the DΨ m differences between cristae and IBM observed using Airyscan imaging, we determined whether the DΨ m would display the same heterogeneity by super-resolution microscopy (e.g., STED). Using living HeLa cells stained with TMRM, we found a nearly identical pattern in the heterogeneity of DΨ m , where, notably, cristae DΨ m significantly exceeds that of IBM (Fig 4G-I).
Altogether, these data demonstrate that the voltage associated with cristae is significantly higher than that of IBM, which is consistent with the higher concentration of ETC components associated with cristae membranes. DΨ m differences between cristae and IBM (DΨ Cr-IBM ) are sensitive to inhibition of F 1 F 0 ATP Synthase and to uncoupling maintain DΨ m that is distinct from the IBM, then inhibiting the consumption of the proton gradient by F 1 F 0 ATP Synthase would further increase the difference between IBM and cristae. To assess this hypothesis, we inhibited F 1 F 0 ATP Synthase with oligomycin and measured the difference in the DΨ m between the cristae and IBM (DΨ Cr-IBM ). We found that not only did oligomycin increase the total DΨ m (Fig 5A and C), as expected (Farkas et al, 1989), but it also increased the DΨ Cr-IBM , hyperpolarizing the cristae (Fig 5B and  C). Conversely, we tested whether treatment with FCCP, a protonophore that shuttles protons across the IMM, would diminish the DΨ Cr-IBM . Consistent with previous studies (Farkas et al, 1989;Loew et al, 1993), we determined that FCCP decreased the total DΨ m ( Firstly, we examined the effect of deleting Mic13 on DΨ Cr-IBM in HeLa cells. While Mic13-KO cells showed a marked decrease in the number of cristae, we identified some regions of IMM that maintained cristae structures (Fig 6A; arrowheads). Mic13-KO mitochondria showed decreased heterogeneity of TMRE staining along the IMM and DΨ Cr-IBM was significantly diminished, compared to control HeLa cells (Fig 6A and B). To further address the role of the MICOS complex in regulating the mitochondrial hetero-potential, we examined cells in which Mic60 or Mic10 were deleted ( Fig EV1C). In these cells, we observed a significant diminishment of heterogeneity of TMRE FI along the IMM (Fig 6C), as well as a drop in DΨ Cr-IBM (Fig 6D and E).
We next studied the effects of Opa1 deletion on DΨ Cr-IBM . Various studies have demonstrated that not only does Opa1 associate with components of the MICOS complex (Barrera et al, 2016;Glytsou et al, 2016), but it appears to have an independent function as a molecular staple, holding the cristae in a closed configuration (Frezza et al, 2006). We hypothesized, therefore, that deletion of ▸ Figure 5. DΨ m differences between cristae and IBM are sensitive to F 1 F 0 ATP Synthase inhibition and uncoupling.
Response of DΨ m to the F 1 F 0 ATP Synthase inhibitor, oligomycin (10 lM), compared to the uncoupler, FCCP (10 lM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE.
A Quantification of total mitochondrial TMRE FI. N = 3 independent experiments. B Quantification of cristae DΨ m relative to IBM (DΨ Cr-IBM ) in response to oligomycin compared to FCCP. F 1 F 0 ATP Synthase is exclusively localized to the cristae and significant increase in DΨ Cr-IBM in response to blocking of F 1 F 0 ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation. N = 3 independent experiments. C Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE. Cells were first stained for 1 h and only then treated with oligomycin or FCCP. Images show hyperpolarization of cristae under oligomycin (middle row; arrowheads) and depolarization of cristae under FCCP (bottom row; arrowheads) compared to control (top row; arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Note that, the DΨ m -independent fraction of TMRE staining is < 5% of the signal in control conditions. D Zoomed-in region of FCCP-treated mitochondria in (C). Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP. These images show diminished heterogeneity of DΨ Cr-IBM values (arrowheads). Scale bar = 500 nm.
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.

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The Opa1 (Fig EV1D), like deletion of components of the MICOS complex, would disrupt the electrochemical boundaries between the cristae and the IBM, thereby equilibrating the potential along the IMM. Our data indicate that deletion of Opa1 indeed results in a significant loss in DΨ Cr-IBM (Fig 7A and B). Altogether, these data indicate that cristae architecture and CJs are essential for the formation of the electrochemical boundaries that allow for DΨ m of cristae to remain different from that of IBM. The role of cristae structure as well as CJ formation and sealing on the generation of the difference in DΨ m between cristae and IBM were studied by disrupting cristae using Mic10-, Mic13-, and Mic60-deficient cells. Mic13 and Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models.
A Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that, TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that DΨ Cr-IBM is decreased ( Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in DΨ m between cristae and IBM. The largest differences in DΨ Cr-IBM arise in cristae vesicles, generated by deleting CJ regulators Intriguingly, in~25% of Mic10 KO cells, we observed structures that appeared to have detached from the IBM and become hyperpolarized ( Fig 6C, lowest row, arrow). To test whether these hyperpolarized structures in Mic10 KO cells had no membrane continuity with the IMM, we performed laser-induced depolarization of mitochondria that included such vesicles. If these hyperpolarized vesicles were unanchored from the IMM, we would expect that they would remain polarized despite the collapse of DΨ m elsewhere in the mitochondrion. Figure 8A shows that such a hyperpolarized structure (arrowhead) maintained its DΨ vesicle for at least 15 s after the rest of the mitochondrion had depolarized. We determined that the DΨ vesicle-IBM of such vesicles was significantly higher than cristae that maintained attachment to the IBM (DΨ Cr-IBM for Control cristae = 10.11 mV vs. DΨ vesicle-IBM for Mic10 KO vesicles = 25.85 mV; P = 0.0005; N = 3 independent experiments). We further hypothesized that deletion of both Opa1 and Drp1 (Fig EV1D)  Hyperpolarized vesicles were quite rare in control cells, suggesting that they are formed in response to bioenergetic dysfunction and/or impaired cristae formation.

Different cristae within a mitochondrion can have different DΨ m , indicating that cristae function as independent bioenergetic units
Previous studies suggest that a single mitochondrion constitutes a single bioenergetic unit (Amchenkova et al, 1988;Skulachev, 2001;Glancy et al, 2015). A typical experiment would involve laserinduced depolarization at one tip of a mitochondrion, which would appear to instantaneously result in a complete collapse of the DΨ m , leading to the conclusion that the entire organelle functions as one electrochemical unit. These time-lapse experiments determined DΨ m at intervals of 5 s or longer after laser-induced depolarization and were employing microscopes that allowed for single mitochondrion, rather single crista, resolution (Amchenkova et al, 1988;Skulachev, 2001). A 5-s interval, however, is enough time to generate and propagate a soluble signaling molecule and/or changes in membrane architecture that could induce depolarization of independent cristae structures in an entire mitochondrion. Thus, to test whether laserinduced depolarization reflected (i) an instantaneous collapse in voltage due to the mitochondrion functioning as an uninterrupted electrochemical conduit; or (ii) a gradual loss of voltage due to a chemical signal and/or structural changes propagating through the organelle, we re-ran the same experiments as previously described but acquired images~20× more rapidly. Visualizing mitochondria from rat myoblasts (L6) using Rho123 (Fig 9A; Movie EV2 shows time series), we exposed a small mitochondrial region (~0.5-1 lm 2 ) to a rapid, high-power pulse of the 2-photon (2-P) laser (arrow, white box). We then observed mitochondria depolarizing in a wavelike (i.e., gradual) manner: The DΨ m -dependent dye dispersed more rapidly near the original site of laser stress, prior to dispersing along areas farther away (arrowheads follow the loss of DΨ m over time). The Rho123 signal is displayed as a multicolored LUT, where pixel intensity is color-coded on a scale where the most- intense pixels appear white (highest DΨ m ) and the least-intense pixels appear blue (lowest DΨ m ). Examining the images immediately following laser-induced depolarization (~0.30,~0.45, and 0.60 s), we found that the mitochondrion loses DΨ m first in proximity to the site of laser stress, because the pixels become more green and blue, whereas the distal site remains polarized, as indicated by the white and red pixels. In Fig 9B, we plotted the pixel intensity of the mitochondrion in 9A as a function of distance from the top to the bottom at different time points. In the image immediately preceding laser-induced depolarization (red line), the peaks and valleys occupy a similar range of intensities from one end of the mitochondrion to the other. Following depolarization induced by the 2-P laser pulse, Rho123 intensity proximal to the site of laser stress drops substantially, while the signal intensity at the distal end remains almost unchanged (green line). These asymmetrical changes in FI are visible at later time points (blue and purple lines), until~2.1 s, when the mitochondrion has lost virtually all of its DΨ m (dark purple line).
In previous studies, laser-induced depolarization experiments were often performed on mitochondria in fibroblasts, which tend to be remarkably elongated. Therefore, fibroblasts represent an optimal system to test the model of the mitochondrion as an "electrical wire" or "power cable", where loss of DΨ m in one place would lead to simultaneous depolarization across the whole organelle (Amchenkova et al, 1988;Skulachev, 2001). Staining mitochondria in human fibroblasts with MTG and TMRE, we performed laserinduced depolarization experiments (Fig 9C). Loading of MTG depends on DΨ m , but it is retained in mitochondria even after depolarization, because, once inside the organelle, it binds covalently to thiol moieties of various proteins (Presley et al, 2003). Partitioning of TMRE to mitochondria, on the other hand, depends entirely on DΨ m . Following 2-P laser pulsation (arrow, white box), there is greater and greater loss of TMRE over time, while the signal from MTG remains. Figure 9D, which shows the TMRE channel alone, demonstrates that, even though the mitochondrion begins to depolarize proximal to the site of the initial laser stress, it maintains its DΨ m , distally, for a period of time. To quantify this asymmetry in DΨ m , we measured TMRE FI immediately after 2-P laser pulsation at sites distal to the initial perturbation (≥ 10 lm from white box) vs. proximal (≤ 1 lm from white box; Fig 9E).
Quantification shows that in the seconds following laser-induced depolarization, mitochondria tend to remain significantly more polarized at sites distant from targeted laser stress, compared to sites close to it. The time gap of seconds for the propagation of the depolarization wave does not support the notion that the IMM functions as a single electrochemical conduit. We next determined the frequency that mitochondria appeared to depolarize in a wavelike vs. instantaneous manner. Time-lapse imaging at approximately 300 ms/frame, we were significantly more likely to observe wavelike instead of instantaneous depolarization (Fig 9F).
Together, using different cell lines and mitochondrial dyes, our data suggest that the DΨ m is organized into multiple, disparate electrochemical domains along the length of the IMM. It further suggests that the laser-induced depolarization phenomenon propagates within the mitochondrion through the diffusion of a signal and/or structural changes rather than by the membrane acting as a continuous electrochemical element.
If cristae possess a measure of functional autonomy, then it is possible that different cristae within a single mitochondrion could maintain significantly different membrane potentials (DΨ Cr ) from each other. To determine whether one crista within a single mitochondrion has higher DΨ Cr compared to a neighboring crista, we first defined the level of stability of DΨ Cr of individual cristae and then asked if the difference between two cristae is larger than the DΨ Cr fluctuation observed over time in a single crista. To explore this possibility, we performed time-lapse imaging of mitochondria stained with MTG and TMRE, and we examined the stability of DΨ Cr generated by different cristae over time. The membrane potential of each crista was calculated by referencing it to the IBM to produce a value of DΨ Cr-IBM . Figure 10A shows an example of four ◀ Figure 9. Laser-induced depolarization results in non-instantaneous loss of DΨ m along the single mitochondrion.
Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy.

A
Representative images of laser-induced depolarization time series from L6 myoblast, stained with DΨ m -dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most-and least-intense DΨ m , respectively. See legend of LUT colors on the right. Note that, at the first time point (~0.15 s), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤ 5 ms), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation ( different cristae-"A-D" (arrowheads)-that we followed for a period of~10 s. Figure 10B shows that the average DΨ Cr-IBM over time of Cristae C and D is more than double that of Cristae A and B, indicating that cristae maintain different membrane potentials. To further examine their relative functional stability over time, we quantified the standard deviation of DΨ Cr-IBM within each crista over time and compared it to the standard deviation of DΨ Cr-IBM between cristae over time. Our data indicate that, over a period of~10 s, there are significantly larger differences between cristae within a single mitochondrion than the variability recorded from any individual crista over time (Fig 10C). To further investigate the extent to which cristae could exhibit functional autonomy, we studied timelapse images of mitochondria stained with MTG and TMRE and discovered instances where mitochondria would undergo partial depolarization (Fig 10D-F; arrowhead, arrows; Movies EV3-EV5 show time series of partial depolarization from E, F). Remarkably, instances of partial depolarization of a single mitochondrion revealed cristae that remained polarized despite the depolarization of adjacent cristae. Analyzing the specific frames showing partial depolarization, we quantified the DΨ Cr-IBM of the polarized cristae vs. depolarized cristae and determined that the polarized cristae maintained a higher DΨ Cr-IBM than neighboring cristae that appeared to have depolarized ( Fig 10G). Altogether, our data demonstrate that cristae display a degree of functional autonomy, highlighting a new role of these critical structures as independent bioenergetic units.

Discussion
DΨ m is the main driving force for proton re-entry through F 1 F 0 ATP Synthase into the mitochondrial matrix (Mitchell, 1961;Mitchell & Moyle, 1969). In this study, we provide evidence, for the first time, that cristae maintain higher DΨ m than IBM and that each individual crista within the same mitochondrion can maintain DΨ m distinct from neighboring cristae. Using the LSM880 with Airyscan and STED microscopy, we directly visualized the relationship of the DΨ m to the IMM in respiring mitochondria. Our observation that cristae and IBM have heterogeneous DΨ m is based on differential partitioning of DΨ m -dependent probes to the different segments of the IMM. We provide several lines of evidence to support that this differential partitioning of the DΨ m -dependent probes is reflecting DΨ m and not an artifact of dye binding to membranes: (i) We repeated the observation with the unique DΨ m -dependent probes, TMRE, TMRM, and Rho123 (Farkas et al, 1989;Loew et al, 1993;Duchen, 2004). If partitioning to cristae were based on non-Nernstian binding, it is unlikely to occur with different dyes. (ii) DΨ Cr-IBM increased in response to oligomycin and decreased in response to FCCP. (iii) Partitioning to the cristae was decreased in models lacking MICOS-complex subunits or Opa1. (iv) Analyzing flickering events, heterogeneity of TMRE FI along the mitochondrion was markedly decreased during instances of depolarization and reestablished following restoration of DΨ m . This confirms that TMRE signal stemming from DΨ m -independent binding of TMRE to the cristae in our experimental system was negligible. (v) The data relating to DΨ Cr-IBM obtained by Airyscan technology were confirmed by STED microscopy. The imaging was performed in different laboratories with different batches of HeLa cells. (vi) We provided data to confirm the same observations in MEFs, HeLa cells, Hap1 cells, L6 myoblasts, H1975 cells, primary mouse hepatocytes, and human fibroblasts. Altogether, this evidence strongly supports the conclusion that cristae and IBM are sufficiently separated, thus being electrochemically insulated from each other, to allow for different membrane potentials to co-exist along the IMM. A limitation of this methodology is the lack of dyes that are completely independent of DΨ m or cardiolipin content. As a result, the NAO and MTG staining of IBM appeared weaker than that of cristae, precluding ratiometric imaging. This limitation only pertains to the differences in DΨ m of cristae vs. IBM but would not affect conclusions related to the DΨ m differences between different cristae within the same mitochondrion. Moreover, the six points listed above support that the TMRE FI differences we observed between cristae and IBM indeed reflect DΨ m differences. After confirming that the heterogeneous partitioning of DΨ mdependent probes was in fact due to differences in DΨ m , we examined the possibility that different cristae within the same ◀ Figure 10. Cristae are unique electrochemical domains, thus constituting independent bioenergetic units.
We calculated the DΨ Cr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging.

A
Stability of cristae DΨ Cr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time.

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The EMBO Journal 38: e101056 | 2019 ª 2019 The Authors mitochondrion could function as unique electrochemical domains and thus as independent bioenergetic units. We observed that, when imaging at high temporal resolution, laser-induced depolarization of elongated mitochondria did not tend to result in a simultaneous collapse of DΨ m across the entire organelle but rather in a rapid, wavelike depolarization. This discrepancy from earlier studies can be attributed to the fact that previous assays involving laser-induced damage did not image the mitochondria rapidly enough after A B C D Figure 11. perturbing a small region of the organelle-for example, acquisition of a single frame expended 5-10 s (Amchenkova et al, 1988;Skulachev, 2001). Critical delays, either in the time it takes to acquire a single frame of a time series (Amchenkova et al, 1988) or in the duration of the perturbation itself (Glancy et al, 2015), will increase the likelihood of missing possible spatial differences in depolarization (originating close to the site of laser-induced damage). Our study suggests that, unless imaging at ≥ 1 frame(s) per second, observing partial depolarization events is improbable. The tendency for one mitochondrion to exhibit localized depolarization indicates that a single organelle does not resemble an electrical wire (Fig 11A  and B) but rather appears to function more like a configuration of interconnected batteries (Fig 11C and D). Our high-or super-resolution imaging of the IMM stained with DΨ m -dependent dyes is consistent with a model of cristae as individual, interconnected batteries. The DΨ m is not homogeneous along the IMM but shows significant differences between the cristae and IBM, demonstrating electrochemical discontinuity between the otherwise physically connected membranes. The relatively more negative DΨ of the cristae membrane is expected to exert a stronger electrical force, keeping protons proximal to the cristae membrane. Indeed, our study can provide an explanation for heterogeneous distribution of protons along the cristae membrane and between the cristae and the IMS Rieger et al, 2014;Pham et al, 2016). Following deletions of various proteins involved in regulating cristae architecture, including CJ formation, we observed a decrease in the difference between the DΨ m at the cristae membranes relative to the IBM. These data suggest that opening of CJs tends to equalize the DΨ m between cristae and IBM by a mechanism that requires further study. The generation of hyperpolarized vesicles, following different genetic perturbations that eliminate CJs, reinforces our general observation that increasing compartmentalization of the IMM, in turn, intensifies the electrochemical gradient. Lastly, our time-lapse imaging experiments show that the DΨ Cr-IBM of an individual crista is less variable over time compared to the DΨ Cr-IBM among different cristae within the same mitochondrion. Furthermore, we observe that, during transient depolarization, some cristae can maintain polarity despite the collapse in DΨ m of adjacent cristae. In summary, our data support a new paradigm where each crista maintains its own DΨ m that is different from both the IBM and from neighboring cristae.
This study raises interesting questions as to why mitochondria organize the DΨ m in this way. One advantage, for example, could be related to the fact that DΨ m constitutes the main energy available to drive protons through F 1 F 0 ATP Synthase to produce ATP. As such, the localization of F 1 F 0 ATP Synthase at the cristae rims appears to be advantageous in terms of proximity to the batteries. Another possible advantage could be compartmentalization of DΨ m in each crista may serve as a safeguard mechanism restricting the impact of localized damage. In the case of the equipotential model, where the inner membrane of the entire mitochondrion represents a single capacitor, a breach in membrane integrity in one crista would cause a collapse in voltage in all cristae and compromise the function of the whole organelle. If, on the other hand, the IMM could maintain numerous, discrete electrochemical gradients, like a group of batteries, then failure of one or more would not invariably jeopardize the entire mitochondrion. This may be of particular relevance in cells harboring a highly interconnected mitochondrial network as opposed to cells with less elongated and/or branched mitochondria. Furthermore, the hetero-potential model suggests that cristae with higher DΨ Cr-IBM could compensate for cristae with impaired function.
Hyperpolarized and depolarized IMM potentials are associated with different states of respiration. While both uncoupling and an increased rate of ATP synthesis dissipate DΨ m , a decrease in ATP synthesis may result in hyperpolarization and increased ROS production. The hetero-potential model of the mitochondrion allows for different cristae to serve different functions. In this model, some cristae could be more dedicated to ATP synthesis, whereas neighboring cristae could play a role in ROS signaling. The hetero-potential model further allows for the consideration that different cristae may engage in primarily complex II vs. complex I respiration, which are associated with different membrane potentials and could be driven by different fuels.
In conclusion, our study identifies a new parameter of mitochondrial function: a mitochondrial hetero-potential arising from the compartmentalization of the DΨ m along the IMM. These findings have wide-ranging implications for human health, because many diseases and medical complications-for example, dominant optic atrophy (Amati-Bonneau et al, 2008;Zanna et al, 2008), ischemia-reperfusion injury (Birk et al, 2013), and even aging (Daum et al, 2013)-are associated with severe perturbations in cristae structure. Pathogenic changes to cristae architecture may undermine the autonomy of cristae bioenergetics, leading to the conversion of the IMM into an equipotential membrane. Consequently, damage inflicted to any isolated region may impact the bioenergetics of the entire mitochondrion, rendering the cell more vulnerable to mitochondrial toxicants and metabolic stress. Restoring the architecture of the IMM, therefore, could represent a viable approach to reestablishing the disparate electrochemical gradients that are the basis for normal mitochondrial function. This study emphasizes the need to directly explore the relationship between cristae structure and function to further elucidate the etiology of such diseases and, in turn, foster more effective therapies for treating them.
◀ Figure 11. Model of cristae as individual bioenergetic units.
A An equipotential representation of the IMM. In this model, the IMM functions as a continuous electrical cable where cristae and IBM have similar DΨ m and where all cristae respond simultaneously to any change in DΨ m occurring in the mitochondrion where they reside. Transition from a hetero-potential to an equipotential, via cleavage of Opa1, for example, could play a role in normal mitochondrial physiology when high conductance and low ATP synthesis are favored, such as thermogenesis or adaptation to excess nutrient. B Cartoon showing IMM as equipotential. C Model of mitochondrial hetero-potential, showing cristae as individual bioenergetic units. Electrochemical autonomy of cristae protects the mitochondrion confronting a bioenergetic crisis, where dysfunction of one or more cristae would not inevitably cause the entire organelle to fail. In this model, cristae and IBM can have different membrane potentials. Closed CJs generate significant differences in charge between the cristae membranes and IBM, which could be related to the concentration of ETC subunits in the lateral side of the cristae and F 1 F 0 ATP Synthase on the cristae rims. D Cartoon showing IMM as hetero-potential.

Live-cell imaging
Cells were plated in CELLview 4-compartment glass-bottom tissue culture dishes (Greiner Bio-One, 627870), PS, 35/10 mm. Dyes (100 nM 10-N-nonyl acridine orange, 15 nM TMRE, 5 lM Rho123, and/or 200 nM MitoTracker Green; Invitrogen) were added to cell culture media and incubated 1-3 h prior to live-cell imaging with the alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objective on the Zeiss LSM 880 with Airyscan. Prior to image analysis, raw .czi files were automatically processed into deconvoluted Airyscan images using the Zen software. HeLa cells imaged with STED were stained with 50 nM TMRM (Invitrogen) for 30 min before imaging. Leica SP8 LSM, fitted with STED module, was used to perform live-cell super-resolution imaging.

Image analysis
Processed Airyscan images were analyzed using ImageJ (Fiji) software. Briefly, prior to cell cropping and quantification, background was subtracted from all images using a rolling ball filter = 50. After developing analysis protocols, we designed macros for high-throughput image quantification and analysis. For representative images in figures, we applied the Window/Level function when demonstrating relevant changes in pixel intensities; when specifically comparing cellular structures, we adjusted pixel intensities to optimally demonstrate relevant changes in structure. Images acquired with STED microscopy were deconvoluted using Huygens deconvolution software.

Statistical analysis
All statistical analyses were performed on GraphPad Prism and Microsoft Excel. Data sets were subjected to D'Agostino-Pearson omnibus and/or Shapiro-Wilk normality tests to assess whether data were normally distributed. Data were subjected to 2-tailed Student's t-tests, and P values < 0.05 were considered statistically significant. Error bars represent SEM, unless otherwise indicated. N = the number of independent experiments. On average,~20 cells were analyzed per independent experiment per condition. Statistical analysis was performed on the averages from independent experiments.
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