L‐OPA1 regulates mitoflash biogenesis independently from membrane fusion

Abstract Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψm) whose origin is unclear. Using structurally distinct genetically encoded pH‐sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed “mitopHlashes”. Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψm drops or respiratory chain inhibition. Opa1 ablation does not alter Δψm fluctuations but drastically decreases the efficiency of mitopHlash/Δψm coupling, which is restored by re‐expressing fusion‐deficient OPA1K301A and preserved in cells lacking the outer‐membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψm uncoupling occurs early during staurosporine‐induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψm by an explosive matrix pH flash.


Referee #2:
The authors present interesting data about mitochondrial flashes that contributes to the understanding of this phenomenon, which is currently not well understood. The data tells a compelling story about the role of matrix pH changes during mitochondrial flashes and how it is connected with mitochondrial membrane potential and requires OPA1 but not mitochondrial fusion. However, the IMS and cristae data is less convincing and would benefit from some additional experimental evidence demonstrating that these probes work. The manuscript is well written and cites/discusses the literature. The manuscript could be improved with the following adjustments: Major Concerns to be addressed: 1. Figure 2A and 3A: To show localization to mitochondria there should be a known mitochondrial marker. While the staining pattern of the images in Figures 2 and 3 appear to be mitochondrial, without comparing it to a known mitochondrial marker it cannot be definitively stated these images demonstrate mitochondrial localization. Therefore, it is recommended that either the wording in the text be changed as these experiments do not verify the localization or that the proper experiments are done with mitochondrial markers to demonstrate the localization (employing the Pearson's correlation coefficient to verify localization).
2. The cristae and IMS probe data suggest that there are too many potential technical issues to state that pH flashes are not detectable in the IMS and the cristae. Given that no treatment was able to demonstrate a change in IMS/cristae pH (including treatments that were expected to alter pH, such as Antimycin A) suggests that these probes are either not sensitive enough and/or not targeted properly, as the authors suggest. The authors should test additional compounds to demonstrate whether IMS or cristae pH changes can be detected with these probes, which would strengthen the conclusion that mitochondrial flashes do not occur in the IMS or cristae. Without additional evidence, I do not think the data supports the conclusion that mitopHlashes are matrix-limited. 3. Figure 5A: Representative images from a control cell should be added to demonstrate the normal diffusion of PA-GFP. Additionally, it does not appear that the same cell is always presented in the representative images at the 1 minute and 60 minute time points. Specifically for Opa1-/-+ WT OPA1, although this could be due to changes in morphology over the assay time. Please confirm that representative images are from the same cell. Figure 7: It would be informative to know what STS does to the matrix pH, in addition to the depolarization rate and the coupling that is presented. Additionally, a 6 hour time point for the western blot analysis should be added to demonstrate cytosolic OPA1 is released with STS treatment. Quantification of the western blot and immunofluorescence should be included to verify the conclusions drawn from these experiments.

4.
Minor Concerns/Comments: 1. Text and Figure legend/panels do not correlated for Figure 2 in a few places on page 6 of the manuscript ( Figure 1F should be changes to 2F; 2F should be changed to 2G), please correct these issues.
2. Figure 3F: The y-axis title presents a ratio of F480/F380, while in Figure 3D the ratio is F488/F405 for the same probe. Confirm whether this is accurate.
3. Figure 5B: In addition to the increase in GFP area, it would be informative to analyze for the decrease in average fluorescence intensity over time. Additionally, please include how many cells were analyzed for each condition from the 3 independent experiments in the figure legend. 4. Page 10: Confirm whether the correct spelling is stauroporin (as written in the text) or stauroporine.
5. Specify pH/ m un/coupling instead of un/coupling alone so not to cause confusion that it could be related to mitochondrial respiration.

Referee #3:
The manuscript by Rosselin et al. is an interesting study that sheds further light onto the significance of mitochondrial flashes. In the present manuscript, the authors make the rather surprising discovery that mitoflashes do not occur in the IMS. They further demonstrate that apoptosis induction leads to an uncoupling between mitoflashes and mitochondrial membrane potential changes. These are two rather important discoveries. The latter part of the paper is currently not well developed and it also appears not well connected to the remainder of the study. Overall, the study is of impressive quality and is based on powerful live cell imaging data. The detailed comments are found below.
1. The abstract is currently rather confusing. While this reviewer agrees with the renaming of mitoflashes, the abstract does not explain this appropriately and the reader is left confused about what are mitophlashes versus mitoflashes.
2. I would like to see some further controls on the different mitochondrial pH indicators. For instance, what happens with membrane permeabilization? Can digitonin be used? In Figure 2F, a ratiometric curve is shown, it should be translated into absolute pH units to get an idea of how much Antimycin A is able to alter the pH.
3. Although the authors have investigated this with matrix probes in their previous paper, an experiment with hyperfused mitochondria should be shown. Is the absence of flashes in the IMS uniform over larger areas? This absence of data is puzzling, since the authors show the Mfn1/2-/control, but not this experiment.
4. Regarding the IMS pH sensors: how was their localization to the IMS verified? Simple IF does not tell much whether they have been correctly targeted. The discussion mentions that there was some mislocalization, but to which extent and how was it determined? Again, the antimycin A experiment lacks the pH units.
5. The STS experiment is interesting, but does not shed much light onto what is the originating cause of the uncoupling between pH and upon apoptosis induction. In their previous paper, the authors have shown that thapsigargin, another apoptosis inducer, did not alter flash frequency. How was this new condition based on STS different from thapsigargin? Would longer exposure with thapsigargin lead to an uncoupling? Is it really the commitment to the death program that is the determinant? A few other death triggers should be looked at to provide insight. 6. Generally speaking: Can the authors connect the STS experiment better to the remainder of the study? For instance, can the authors connect their observations to the Opa1 status, its oligomerization or its localization? The authors mention that coupling does not depend on Opa1 cleavage, but what happens to it upon apoptosis?
Minor points: 1. On page 6, Figures 1F and S1 are mentioned. Where are they? 2. The text regarding Figure 5 is confusing. The concept of Mfn1/2-/-is introduced, but not appropriately separated from the further Opa1 experiments.
3. The title of the results text describing Figure 6 is confusing. It states that long, uncleaved Opa1 mediates coupling, but in the paragraph, it is concluded that Opa1 processing is dispensable for mitoflash generation. Do the authors simply mean "Both OPA1 variants can mediate coupling"? The comments of the reviewers are in italics, our responses to these comments are in plain letters.

Referee #1:
The manuscript by Rosselin  We thank the reviewer for the constructive comments. We have addressed the issues raised experimentally, and the new data strengthen our initial findings on the role of OPA1 in mitopHlash biogenesis. A point-by-point answer to the reviewer queries is provided below.
Specific Comments: 1. Authors showed mitochondrial localization of pH-probes using confocal microscopy ( Figure 2, 3), indeed they are showing typical mitochondrial localization. However, authors should show their co-localization with already established mitochondrial markers to rule out any ambiguity.
As requested we have confirmed the mitochondrial localization of the probes using antibodies against-Hsp60 as an endogenous marker. As shown in supplementary figure EV1, all the probes colocalized with Hsp60 with Pearson and Mander's coefficients around 80%, thereby establishing their mitochondrial localization. In addition, we have performed immuno-EM with anti-GFP antibodies to precisely assess the localisation of our GFP-based pH probes in different mitochondrial compartments. These new data show that CIV8-spHlu is preferentially detected in cristae while MPP-spHlu is mainly detected in the matrix, as predicted from their targeting sequences. These data firmly establish the correct localization of the probes and are now included as Figure 3 (new).

Authors showed that OPA1 ablation did not affect the ΔΨm, instead alters the mitoflash/ΔΨm coupling. However, in their earlier study (Santo-Domingo et al 2013) they claim that OPA1 is necessary for mitoflash generation and "not a single pHmito flash was detected in Opa1-/-cells even after application of atractyloside".
To contrast the earlier study from same group, in current study they found that "Using TMRM spikes as indicators of activity, they could then detect residual mitopHlashes events in Opa1-/-cells, indicating that Opa1 ablation severely impairs, but does not abrogate mitopHlash activity. Please elaborate this discrepancy.
Discussed together with point 3 below.
The detection of transient depolarization and mitopHlashes is challenging in Opa1 -/cells whose mitochondria are totally fragmented and mobile. Imaging these stochastic transients requires a microscope with a good signal-to-noise ratio and an excellent resolution. The discrepancy between the two studies likely stems from the different sensitivity of the microscopes used to record mitoflashes. In our earlier publication, the experiments were performed on a spinning disk microscope while we used here a Nikon A1r laser scanning confocal microscope. The increased sensitivity of the newer microscope facilitated the visualization of both the TMRM and SypHer events, revealing the residual activity of Opa1 -/cells. A representative experiment illustrating the challenge posed by the detection of sporadic depolarization transients occurring randomly in time and space within interconnected mitochondria is now shown in Fig EV5 and movie EV3. Science is a self-correcting endeavour and while we apologize for the confusion that the discrepancy with our earlier study might create, we believe that our newer and more accurate findings in fact strengthen and refine the conclusion that OPA1 plays an important role in mitopHlashes biogenesis. In support for our new findings, a recent study also reported residual mitopHlases in OPA1-null cells, the low frequency of these residuals increasing upon osmotic challenge [1].

Authors claim that OPA1 stabilizes the respiratory complexes active conformation, which enables respiring mitochondria to pH/ΔΨm coupling. To validate this hypothesis authors should give biochemical experimental evidence for the involvement of L-OPA1 and fusion deficient OPA1 mutant in RCS assembly/disassembly.
We have attempted to quantify the formation of supercomplexes in the different cell lines on blue native gels, but despite our efforts failed to obtain reproducible results. These experiments are notoriously difficult and although we might be able to obtain valid data by refining the experimental conditions, we believe that the expected outcome of these biochemical experiments does not warrant the time, effort, and financial investment required. Instead, we have softened our conclusion and now only present supercomplex stabilization as one possible mechanism of the OPA1 action.

Page 7: Although authors conducted an experiment using oligomycin and galactose rich media which does not have any effect on cristae or IMS pH, it is worth included as a supplementary information.
We now include these experiments as Fig EV3. No pH change was detected with the pHluorin probes when oligomycin was added to the imaging media. A representative experiment conducted with Smac-rpHluorin is shown (Fig EV3E). When HeLa cells were grown in galactose media for 48h, no mitoflash was detected during membrane depolarization using the probes smac-rpHluorin, CVe-spHluorin and CIV8-spHluorin ( Fig EV3G, representative experiment using CIV8-spHluorin). However a significant change in resting pH was observed compared to low glucose media ( Fig  EV3F), consistent with an earlier study [2]. Fig. 6E missing from the text. The authors present interesting data about mitochondrial flashes that contributes to the understanding of this phenomenon, which is currently not well understood. The data tells a compelling story about the role of matrix pH changes during mitochondrial flashes and how it is connected with mitochondrial membrane potential and requires OPA1 but not mitochondrial fusion. However, the IMS and cristae data is less convincing and would benefit from some additional experimental evidence demonstrating that these probes work. The manuscript is well written and cites/discusses the literature. The manuscript could be improved with the following adjustments:

Description of
We thank the reviewer for the constructive comments. We have addressed the issues raised experimentally, and the new data confirm the correct localization of the IMS and cristae probes. A point-by-point answer to the reviewer queries is provided below.
Major Concerns to be addressed: 1. Figure 2A  We have confirmed the mitochondrial localization of the probes using antibodies against-Hsp60 as an endogenous marker. All the probes co-localized with Hsp60 with Pearson and Mander's coefficients around 80% (Fig EV1), thereby establishing their mitochondrial localization. We have also performed immuno-EM with anti-GFP antibodies to precisely assess the localisation of our GFP-based pH probes in different mitochondrial compartments. These new data show that CIV8-spHlu is preferentially detected in cristae while MPP-spHlu is mainly detected in the matrix, as predicted from their targeting sequences. These data firmly establish the correct localization of the probes and are now included as Figure 3 (new). We now show that the cristae probe CVe-spHluorin reports different pH values when HeLa cells are grown in culture media permissive for oxidative phosphorylation or for glycolysis ( Fig EV3F). Consistent with a previous study [2], we measured a resting pH of 7.2 in cells grown in 10 mM galactose and 7.0 in 5.5 mM glucose, an acidification that likely reflects the reverse activity of the ATP synthase in 5.5. mM glucose [3]. Together with our immuno-EM data (Fig 3) and the response of IMS and cristae probes to H+ ionophores (Fig 3), this demonstrates that our probes targeted to the IMS and cristae report acute and chronic changes in the pH of intra-mitochondrial compartments.

The cristae and IMS
We have tested a battery of pharmacological tools and failed to detect significant changes in IMS and cristae pH during acute inhibition of respiratory complexes. Without time-resolved control recordings of pH changes in IMS and cristae during acute RCS inhibition, we cannot rule out that mitopHlashes might propagate to these compartments without causing a detectable signal, although this appears unlikely given the amplitude of the concomitant matrix pH response. We have rephrased the text of our manuscript accordingly.
3. Figure 5A: Representative images from a control cell should be added to demonstrate the normal diffusion of PA-GFP. Additionally, it does not appear that the same cell is always presented in the representative images at the 1 minute and 60 minute time points. Specifically for Opa1-/-+ WT OPA1, although this could be due to changes in morphology over the assay time. Please confirm that representative images are from the same cell.
We have added pictures of a WT MEF cell in Figure 5A. The images taken at 1 and 60 minutes are from the same cell for each condition and this is now specified in the figure legend and in the material and methods section. Good point, because the mitopHlash frequency is affected by changes in the matrix pH. We have verified that the matrix pH remained stable for up to 2h during STS treatment. The data are shown below for the reviewer perusal and the pH values indicated in the text (page 11, line 4).
Effect of STS on matrix pH. Matrix pH was measured as described in material and methods in MEF cells expressing mito-sypHer and exposed to DMSO or I µM STS for 2 h. Images were acquired on a Nikon A1r inverted confocal microscope. Values are mean±SD of 3 independent experiments.
We now include an 8h time point demonstrating that OPA1 is released in the cytosol following STS treatment ( Fig 7C) and have quantified the extent of OPA1 and cytochrome c release (Fig 7C and  7D).
Minor Concerns/Comments: Figure 2 in a few places on page 6 of the manuscript ( Figure 1F should be changes to 2F; 2F should be changed to 2G), please correct these issues.

Text and Figure legend/panels do not correlate for
Corrected, thank you.
2. Figure 3F: The y-axis title presents a ratio of F480/F380, while in Figure 3D the ratio is F488/F405 for the same probe. Confirm whether this is accurate.
The values are correct. A confocal microscope was used for the pH calibration shown in Fig 3D and a wide-field microscope for the time-resolved recordings shown in Fig 3F, using UV illumination matching the excitation peak of the probe.

Figure 5B: In addition to the increase in GFP area, it would be informative to analyze for the decrease in average fluorescence intensity over time. Additionally, please include how many cells were analyzed for each condition from the 3 independent experiments in the figure legend.
We have measured the decrease in PA-GFP fluorescence over time. These data are shown below for the reviewer perusal. The loss of PA-GFP fluorescence was more pronounced in WT and Opa1 rescued KO cells after 60 min, but the differences are not significant and we did not include these data in the revised MS. The number of cells analysed for each condition is now included in the figure legend.

Page 10: Confirm whether the correct spelling is stauroporin (as written in the text) or stauroporine.
Corrected (staurosporine).

Specify pH/ΔΨm un/coupling instead of un/coupling alone so not to cause confusion that it could be related to mitochondrial respiration.
Corrected.

Referee #3:
The manuscript by Rosselin et al. is an interesting study that sheds further light onto the significance of mitochondrial flashes. In the present manuscript, the authors make the rather surprising discovery that mitoflashes do not occur in the IMS. They further demonstrate that apoptosis induction leads to an uncoupling between mitoflashes and mitochondrial membrane potential changes. These are two rather important discoveries. The latter part of the paper is currently not well developed and it also appears not well connected to the remainder of the study. Overall, the study is of impressive quality and is based on powerful live cell imaging data. The detailed comments are found below.
We thank the reviewer for the constructive comments and provide a point by point answer to the detailed comments below.

The abstract is currently rather confusing. While this reviewer agrees with the renaming of mitoflashes, the abstract does not explain this appropriately and the reader is left confused about what are mitophlashes versus mitoflashes.
We now explain the new terminology in the abstract. Figure 2F, a ratiometric curve is shown, it should be translated into absolute pH units to get an idea of how much Antimycin A is able to alter the pH.

I would like to see some further controls on the different mitochondrial pH indicators. For instance, what happens with membrane permeabilization? Can digitonin be used? In
We now provide recordings translated into absolute pH values to illustrate the effects of Antimycin A in Fig 2F and Fig EV3. We also show that the cristae probe CVe-spHluorin reports different pH values when HeLa cells are grown in culture media permissive for oxidative phosphorylation or for glycolysis ( Fig EV3F). Consistent with a previous study [2], we measured a resting pH of 7.2 in cells grown in 10 mM galactose and 7.0 in 5.5 mM glucose, an acidification that likely reflects the reverse activity of the ATP synthase in 5.5. mM glucose [3]. Together with our immuno-EM data (Fig 3) and the response of IMS and cristae probes to H+ ionophores (Fig 2), this demonstrates that our probes targeted to the IMS and cristae report acute and chronic changes in the pH of intramitochondrial compartments. We had previously documented the effect of respiratory chain inhibitors on the matrix mitopHlashes [4]. None of these inhibitors evoked significant acute changes in IMS or cristae pH when applied (Fig. EV3C-E), and flashing events were not detected in the cristae of cells cultured in galactose-rich media (Fig. EV3G). As discussed in the response to reviewer 2, without time-resolved control recordings of pH changes in IMS and cristae during acute RCS inhibition, we cannot rule out that mitopHlashes might propagate to these compartments without causing a detectable signal, although this appears unlikely given the amplitude of the concomitant matrix pH response. The effects of acute addition of 100 µM digitonin are shown below: Upon digitonin addition, mitochondria fragmented and the fluorescence of the matrix and cristae probes became sensitive to imposed changes in the pH of the extracellular solution. This is consistent with our earlier observation that cytosolic pH changes rapidly propagate to the mitochondrial matrix [5].

Although the authors have investigated this with matrix probes in their previous paper, an experiment with hyperfused mitochondria should be shown. Is the absence of flashes in the IMS uniform over larger areas? This absence of data is puzzling, since the authors show the Mfn1/2-/control, but not this experiment.
As suggested, we have measured pH changes in the IMS and cristae of cells expressing a dominantnegative DRP1 to enforce mitochondrial fusion. The mitochondrial area exhibiting stochastic drops in DPM measured with TMRM was greatly increased in cells with hyperfused mitochondria ( Fig  EV4), but pH flashes remained undetectable with smac-rpHluorin, CIV8-spHluorin and CVe-pHluorin. We also used hyperosmotic stress conditions known to increase flash frequency [6] but failed to detect pH flashes with the probes targeted to the cristae or the IMS (data not shown).

Regarding the IMS pH sensors: how was their localization to the IMS verified? Simple IF does not tell much whether they have been correctly targeted. The discussion mentions that there was some mislocalization, but to which extent and how was it determined? Again, the antimycin A experiment lacks the pH units.
We now show that all our probes co-localize with the mitochondrial marker Hsp60 (Fig EV2) and have quantified the distribution of a cristae and matrix probe in mitochondrial subcompartments by immunogold electron microscopy (Fig 3, new). As expected, CIV8-spHluorin immunoreactivity was mainly detected in the cristae and the IMS, the residual immunoreactivity detected in the matrix and outside mitochondria reflecting either mislocalization or non-specific binding. The antimycin A recording is now reported in pH units.

The STS experiment is interesting, but does not shed much light onto what is the originating cause of the uncoupling between pH and ΔΨ upon apoptosis induction. In their previous paper, the authors have shown that thapsigargin, another apoptosis inducer, did not alter flash frequency.
How was this new condition based on STS different from thapsigargin? Would longer exposure with thapsigargin lead to an uncoupling? Is it really the commitment to the death program that is the determinant? A few other death triggers should be looked at to provide insight.
We previously used thapsigargin to study the effect of ER Ca 2+ store depletion on mitopHlashes, a process developing on a time scale too short to induce detectable apoptosis (8 min vs. 2h). As suggested, we have analysed the effect of two other death triggers, H 2 O 2 and etoposide. Our new data show that these two agents induce a significant decrease in pH/ΔΨm coupling (Fig EV5A and  EV5B). We thank the reviewer for suggesting this experiment.

Generally speaking: Can the authors connect the STS experiment better to the remainder of the study? For instance, can the authors connect their observations to the Opa1 status, its oligomerization or its localization? The authors mention that coupling does not depend on Opa1 cleavage, but what happens to it upon apoptosis?
We now show that pH/Dy m uncoupling is associated with the release of OPA1 in the cytosol ( Fig  7C). The intensity of the OPA1 reactive band detected in the cytosolic fraction progressively increased after 2h and 8h of staurosporine exposure.
Minor points: 1. On page 6, Figures 1F and S1 are mentioned. Where are they?
Corrected, thank you. Figure 5 is confusing. The concept of Mfn1/2-/-is introduced, but not appropriately separated from the further Opa1 experiments.

The text regarding
We have rephrased this section to better integrate the Mfn1/2 -/experiment with the general context of our study. Figure 6 is confusing. It states that long, uncleaved Opa1 mediates coupling, but in the paragraph, it is concluded that Opa1 processing is dispensable for mitoflash generation. Do the authors simply mean "Both OPA1 variants can mediate coupling"? 6E shows that the proteolytic processing of OPA1 is not required for pH/ΔΨm coupling. This implies that the long, uncleaved OPA1 forms are sufficient to mediate coupling since in the absence of the Oma1 and Yme1 proteases only the long forms or OPA1 remain. Fig. 6B shows that either the V1 or the V7 variant can reconstitute coupling in Opa1 null cells, implying that the two variants are redundant for this function. The logical conclusion is, as the reviewer suggests, that both OPA1 variants can mediate coupling, without requiring processing. We changed the title of Thank you for the submission of your revised manuscript to EMBO reports. I apologize for the delay in getting back to you with a decision on your manuscript. We were still hoping to receive feedback from referee 2 on it, but despite serious efforts from our side, this reviewer has not submitted his/her review. Therefore, I will make a decision based on the two reports we have received so far and which are both positive. Moreover, both referees indicated that the concerns of referee 2 have been adequately addressed in their opinion.

The title of the results text describing
As you will see, referee 3 suggests some minor changes that need to be addressed before we can accept your manuscript for publication. From the editorial side, there are also a few things that we need before we can proceed with the official acceptance of your study. -

Referee #1:
The results and the data in the revised manuscript has been significantly improved. All the reviewer's concerns were thoroughly addressed. It is worth publishing in the EMBO Repots.

Referee #3:
The authors have addressed all comments by myself and the other reviewers adequately. The manuscript is now of very high technical quality. This is a very nice work that will be well respected within the community. Very few minor changes or suggestions should be considered: 1. The contemporaneous nature of pH and changes should be shown in an aligned, zoomed-in way in Figure 1H, where we can see whether the two are perfectly aligned or whether there is maybe a small time difference between the two readouts. Were any differences in the exact time points of rises/falls of the two measurements EVER found?
2. The graph in Figure 4C appears to not be representative, when compared to 4E.
2nd Revision -authors' response 19 December 2016 Referee #1: The results and the data in the revised manuscript has been significantly improved. All the reviewer's concerns were thoroughly addressed. It is worth publishing in the EMBO Repots.
We thank the reviewer for the positive comments. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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Corresponding Author Name: Nicolas Demaurex