MTCH2 cooperates with MFN2 and lysophosphatidic acid synthesis to sustain mitochondrial fusion

  1. Andres Goldman
  2. Michael Mullokandov
  3. Yehudit Zaltsman
  4. Limor Regev
  5. Smadar Levin-Zaidman
  6. Atan Gross

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Abstract

Fusion of the outer mitochondrial membrane (OMM) is regulated by mitofusin 1 (MFN1) and 2 (MFN2), yet the differential contribution of each of these proteins is less understood. Mitochondrial carrier homolog 2 (MTCH2) also plays a role in mitochondrial fusion, but its exact function remains unresolved. MTCH2 overexpression enforces MFN2-independent mitochondrial fusion, proposedly by modulating the phospholipid lysophosphatidic acid (LPA), which is synthesized by glycerol-phosphate acyl transferases (GPATs) in the endoplasmic reticulum (ER) and the OMM. Here we report that MTCH2 requires MFN1 to enforce mitochondrial fusion and that fragmentation caused by loss of MTCH2 can be specifically counterbalanced by overexpression of MFN2 but not MFN1, partially independent of its GTPase activity and mitochondrial localization. Pharmacological inhibition of GPATs (GPATi) or silencing ER-resident GPATs suppresses MFN2’s ability to compensate for the loss of MTCH2. Loss of either MTCH2, MFN2, or GPATi does not impair stress-induced mitochondrial fusion, whereas the combined loss of MTCH2 and GPATi or the combined loss of MTCH2 and MFN2 does. Taken together, we unmask two cooperative mechanisms that sustain mitochondrial fusion.

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  1. Version published to 10.1038/s44319-023-00009-1
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    Reply to the reviewers

    Reviewer #1

    1-1) Authors conclude that "MFN2 and MTCH2 compensate for each other's absence", but the compensation works only when they are overexpressed. On the other hand, upon HBSS or CHX treatment, MFN2 KO or MTCH2 KO cells have elongated/hyperfused mitochondria, but this is not observed in double deficient cells. In this case, MFN2 and MTCH2 show compensatory effects on mitochondria elongation. The authors believe the two conditions, unstressed & OE and stressed conditions, activating same molecular machineries, but it is not fully supported by the data. For example, in unstressed condition, MTCH2 OE can recover MFN2 KO but not of MFN1 KO, suggesting MTCH2-dependent mitochondria fusion requires MFN1, but it is not tested for stressed condition.

    Furthermore, even if the machineries are common among stressed and unstressed conditions, authors should discuss why the endogenous MFN2/MTCH2 expression is not enough to activate mitochondria fusion in MTCH2/MFN2 KO cells, respectively, and how the compensatory effects are activated upon HBSS or CHX treatment.

    __R: We thank the reviewer for this important comment. __

    1) The reviewer’s comment is correct; in resting conditions the endogenous expression of MFN2 in MTCH2 KO and vice-versa are not sufficient to compensate for each other’s absence, since in steady state they both remain largely fragmented. Thus, we corrected the text accordingly by removing this concept. Interestingly, in the revised MS we show that MFN2 KO results in an increase in MTCH2 expression levels in the mitochondria and to a strong decrease in the GPAT3 and 4 expression levels in the ER (Rev Fig. 3B, C). These results suggest that: 1) Expression of MFN2 is important for the stabilization of GPAT3 and 4 (perhaps they form a functional complex together); 2) the expression levels of endogenous MTCH2 are possibly elevated to compensate for the decreased biosynthesis of LPA and increased demand of LPA funneling for mitochondrial fusion. This hypothesis was added to the discussion.

    2) In the revised MS, we tested the possibility that MTCH2 overexpression together with mitochondrial fusion stress could enforce mitochondrial fusion in MFN1 KO cells, and found that it could not (Rev Supp Fig. 4 J-K).

    3) We have also discussed what might be the compensatory effects of MTCH2 and MFN2 when activated upon enforced mitochondrial fusion induced by protein overexpression and stress (page 20 last paragraph).

    1-2) Authors found out that ER-targeted MFN2 can rescue the mitochondria fragmentation in MTCH2 KO MEF cells, but mitochondria-targeted MFN2 has a lower effect than Wt MFN2. (Fig 2A&B). This finding suggests that MTCH2 loss might impair MFN2 localization at the ER. The authors should investigate endogenous MFN2 localization in MTCH2 KO MEFs.

    R: We thank the reviewer for this insightful comment. We addressed this point and show that MTCH2 deletion does not change the expression levels or the subcellular localization of endogenous MFN2 (Rev Fig. 3B).

    1-3) The analysis to test whether recovery of mitochondrial morphology by ER-targeted MFN2 in MTCH2 KO depends on LPA synthesis or not is missing (Fig 3G). Authors should examine whether mitochondrial elongation induced by ER-localized MFN2 in MTCH2 KO cells is impaired by the GPAT inhibitor.

    R: This analysis was performed (we also included the other mutants), and presented the new data in the revised MS (Rev Supp Fig. 3N, O).

    1-4) In the discussion section, authors suggest that mitochondrial LPA would be a crucial factor for MFN2 dependent mitochondrial fusion. To test this hypothesis, authors should overexpress mitochondrial GPAT and evaluate its effect on mitochondrial morphology.

    R: We thank the reviewer for this important suggestion. We ordered two commercially available plasmids encoding GPAT1 to address this point (DNASU ____HsCD00082324 and HsCD00082324)____ but unfortunately the proteins were not expressed in our cells. In addition, we revised the text and changed the angle of the interpretation of these results, and clarified that inhibiting LPA impairs MTCH2-independent mitochondrial plasticity in response to MFN2 overexpression, rather than MFN2-dependent mitochondrial fusion.

    1-5) In the discussion section, authors indicated that ER-targeted MFN2 could recover mito-ER contacts leading to LPA flux from ER to mitochondria and mitochondria elongation in MTCH2 KO. However, MTCH2 KO itself already have more mito-ER contacts (Fig 2D-H), and an artificial linker fails to recover mitochondria fragmentation in MTCH2 KO cells (Fig S2C, D). Thus, increased number of contacts appears not sufficient to recover the phenotype. The authors should consider this point in the discussion.

    R: We added a comment on this important point in the results section (page 10, line 9)

    1. Certain methods are not appropriate to support the stated conclusions.

    2-1) Authors assess "mitochondria fusion" by evaluating mitochondrial morphology. The authors also describe mitochondrial clumping as a fusion-impaired phonotype (Fig 4A&B). Mitochondrial fusion should be evaluated using a PEG assay or a mtPA-GFP analysis.

    R: We now provide in the revised MS results of a mtPA-GFP analysis done for MTCH2 KO MEFs exposed to GPATs inhibitor and treated with CHX (Rev Fig. 4G, H). This experiment supports the notion that loss of MTCH2 along with LPA synthesis inhibition largely impairs mitochondrial fusion in response to CHX.

    2-2) In figure 2D-G, authors show that MTCH2 KO cells have more and longer mitochondria-ER contacts. The correct experiment is not to compare these cells to WT, but to KO reconstituted with MTCH2.

    R: The reviewer is correct however it will take us many more months to generate a stable MTCH2 rescue cell line and to perform EM analysis of these cells, which would significantly slow down the revision of our MS.

    __ __2-3) Since staining of MitoTracker depends on mitochondrial membrane potential, mitochondria with low potential would be invisible and excluded from the analysis. Authors should investigate mitochondrial morphology by immunostaining also in Fig 4D, S4A, D, and K.

    __R: We agree with the reviewer’s comment, ____but re-doing all these experiments will be too labor-intensive and time consuming. We therefore focused on Supp Fig. 4A, in which the combination of FSG67 (GPATi in the revised MS) and HBSS treatment impaired mitochondrial membrane potential and mitochondria did not uptake the MitoTracker dye. We repeated this experiment using immunofluorescence, performed new quantifications, and incorporated the new data into the MS (Rev Fig. 4A). We did not remove Supp Fig. 4A since we wanted to emphasize the point that the combination of LPA synthesis inhibition and amino-acid deprivation results in loss of mitochondrial membrane potential. __

    Minor comments

    1. Since authors use FSG67, an inhibitor against GPAT1, 2 and 3, knocking down of each of GPATs will improve the significance of this work.

    __ R: We thank the reviewer for suggesting these experiments. Since the contribution of mitochondrial GPATs to mitochondrial fusion was already established, we complemented our studies by silencing ER GPATs 3 and 4. We tested the contribution of ER-GPATs to MTCH2-independent mitochondrial fusion elicited by MFN2 overexpression (Rev Fig. 3L-N) and induced by either HBSS or CHX (Rev Fig. 4I-K).__

    1. Recently, a paper about MTCH2 is published (Guna et al., Science), which shows its insertase activity on tail anchored proteins. Authors should include this point of view in the discussion.

    R: We performed new experiments to address this important point. We evaluated the effect of MTCH2 deletion on the expression and localization of the fusion and fission proteins and on the LPA synthesis proteins, and found minor changes (Rev Supp Fig. 1D and Rev Fig. 3B, C). Thus, the effects we are seeing in the MTCH2 KO cells do not seem to be directly related to its insertase activity.

    3-1) There are some discrepancies with the previous Labbé et al article.: Labbé et al. suggest that MTCH2 activity on mitochondria (from HCT116 cells) fusion is dependent on LPA, based on in vitro fusion assay. On the other hand, this manuscript shows that inhibition of LPA synthesis could not block MTCH2-induced mitochondria elongation in WT MEF and HEK293T cells. MTCH2 KO HCT116 cells are resistant to HBSS- but sensitive to CHX-induced mitochondria elongation. In this manuscript, MTCH2 KO MEF cells are sensitive to both stimuli, and only when MTCH2- and MFN2-deficient MEF cells are resistant to both stimuli. These discrepancies would be caused by difference of assay system or cell lines, but it is not clearly addressed.

    R: We thank the reviewer for raising these discrepancies. ____Despite the discrepancies in the response of MTCH2 KO MEFs to HBSS, both manuscripts largely support the model that MTCH2 funnels LPA towards mitochondrial fusion sites. Our data suggests that loss of MTCH2 unmasks the requirement of sufficient LPA synthesis to sustain mitochondrial fusion, and we also show that MTCH2 overexpression can enforce mitochondrial fusion in the presence of GPATs inhibitor. These two results can be conciliated by the model that MTCH2 is able to optimize or funnel the levels of LPA towards the mitochondrial fusion sites, and when MTCH2 is absent and not able to catalyze this process, mitochondria rely on LPA levels to stay elevated to enable mitochondrial fusion but in a less efficient way. Interestingly, since we show that mitochondrial fusion enforced by HBSS shows full dependency on LPA synthesis, it is expected that loss of MTCH2 will have a stronger impact on HBSS-mediated mitochondrial fusion than on CHX. Nevertheless, MEFs clearly and consistently are sensitive to HBSS, yet this does not exclude that MTCH2 deletion in combination with HBSS treatment may be synergistically detrimental for the cell in other aspects of its well-functioning.

    Reviewer #2

    Specific points

    The authors should comment on recently published work in Science that MTCH1 and likely MTCH2 are outer membrane insertases. The data in the manuscript seem consistent with the model that an undetermined protein may be poorly inserted in a MTCH2 knockout leading to reduced fusion activity mediated by MFN1. Could this explain how MTCH2 overexpression selectively restores fusion to MFN2 KO cells?

    R: We thank the reviewer for raising this important point, and we have now performed new experiments to address it. We evaluated the effect of MTCH2 deletion on the expression and localization of the fusion and fission proteins and on the LPA synthesis proteins, and found minor changes (Rev Supp Fig. 1D and Rev Fig. 3B, C). Thus, the effects we are seeing in the MTCH2 KO cells do not seem to be directly related to its insertase activity.

    The authors make the claim that "MFN2 and MTCH2 compensate for each other's absence" though this is not supported by their data. For example, MFN2 expression is not affected in an MTCH2 KO but cannot compensate to promote fusion. Rather, the authors find that MTCH2 and MFN2 are capable of promoting fusion when overexpressed in the absence of the other.

    __R: We thank the reviewer for this important comment. __

    The reviewer’s comment is correct, and in resting conditions the endogenous expression of MFN2 in MTCH2 KO cells and vice-versa are not sufficient to compensate for each other’s absence, since in steady state they both remain largely fragmented. Thus, we corrected the text accordingly by removing this concept. Interestingly, in the revised MS we show that MFN2 KO results in an increase in MTCH2 expression levels in the mitochondria and to a strong decrease in the GPAT3 and 4 expression levels in the ER (Rev Fig. 3B, C). These results suggest that: 1) Expression of MFN2 is important for the stabilization of GPAT3 and 4 (perhaps they form a functional complex together); 2) the expression levels of endogenous MTCH2 are possibly elevated to compensate for the decreased biosynthesis of LPA and increased demand of LPA funneling for mitochondrial fusion. This hypothesis was added to the discussion.

    The authors rely heavily on claims that tagged MFN1 and MFN2 are fully functional, but is it possible that MFN1-GFP is only partially functional? Does untagged MFN1 overexpression cause mitochondrial fusion in a MTCH2 KO? The quantification of this is done only with aspect ratio, and not by categorization of mitochondrial morphology (Fig. S1). The authors should present both analyses in this and all other experiments in the manuscript, particularly since aspect ratio is only performed on 15 cells per condition and not on experimental replicates. The authors should clarify if sample identity was blinded prior to analysis.

    __R: As suggested by the reviewer, we repeated all the MFN1 overexpression experiments using an untagged version of the protein, which was overexpressed in MFN1 KO, MTCH2 KO and MFN2 KO MEFs. The new data was included in Rev Fig. 1C-E. The data is consistent with our previous observations using MFN1-GFP. Also, mitochondrial morphology classification was added to the majority of the experiments presented in our MS, which represents quantification of three separate technical repetitions of the experiments (this analysis was not performed blinded). __

    The constructs that form the basis of conclusions of ER versus mitochondrial-targeted MFN2 require additional controls to support robust conclusions. The immunofluorescence data suggests that MFN2-ACTA to some degree targets outside of mitochondria (Fig. 2A), and also that MFN2-YIFFT seems to localize to some degree to mitochondria (Fig. 2A). The YIFFT construct may also localize more prevalently to mitochondria in the presence of ACTA (Fig. S2A). Mistargeting would make interpretation of these experiments not possible, as these constructs must exclusively localize to their intended organelle to make strong conclusions. Triple labeling with ER and mitochondrial markers would be helpful, as well as western blots to confirm consistent expression levels and protein stability of each construct. The ACTA MFN2 also appears to promote fusion in a MTCH2 KO. How is this reconciled with the conclusion that ER-targeting of MFN2 is required?

    R: We thank the reviewer for these important concerns. We repeated all the MFN2 mutant experiments, including the GTPase mutant MFN2 K109A, and included ER labelling to the MFN2-IYFFT and MFN2-ACTA transfections (Rev Fig. 2B; Supp Fig. 2A, C, D). We also included line fluorescence plot analysis to show localization of MFN2 mutants along with ER or mitochondrial markers (Rev Fig. 2C). Because these studies are cell-based analysis, which were performed using transient transfection, and the efficiency of expression of some of these constructs was very low, it would be technically very difficult to detect their expression using subcellular fractionation. Nevertheless, the constructs MFN2-IYFFT-FLAG and MFN2-ACTA used in our MS were previously reported and their subcellular localization was confirmed (____Sugiura, A. et al. (2013) ‘MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2’, Molecular Cell, 51(1), pp. 20–34. doi:https://doi.org/10.1016/j.molcel.2013.04.023)____.

    The authors conclude that "MFN2-mediated fusion requires LPA synthesis", but what is shown instead is that GPATi is epistatic to fusion caused by overexpression of MFN2. The authors should be careful about drawing strong conclusions from their overexpression studies. While MTCH2 overexpression causes hyperfusion in the presence of GPATi, this does not mean that LPA doesn't promote MTCH2-dependent fusion, merely that GPATi does not block hyperfusion caused by MTCH2 overexpression.

    R: We thank the reviewer for pointing out these issues. We have revised the text accordingly, and instead of "MFN2-mediated fusion requires LPA synthesis" we wrote “GPATi is epistatic to fusion caused by overexpression of MFN2”__ (page 13, line 14). ____We also added “____reducing LPA levels by inhibiting GPAT activity is insufficient to impair mitochondrial fusion enforced by MTCH2-overxpression____”__ (page 13, line 9).

    The collapse of the mitochondrial network in MTCH2 KO cells treated with cycloheximide + GPATi does not indicate the cells "require newly-synthesized LPA to respond to SIMH". Instead, it suggests that mitochondria in MTCH2 KO cells are sensitized to combined GPATi/cycloheximide treatment. Could this collapse be fusion-independent? If mitochondria are treated with nocodazole to relax the mitochondrial network (as in Smirnova et al, MBoC, 2001 or Yang et al, Nat Comm, 2022), does the mitochondrial network appear hyperfused?

    R: Thank you and as the reviewer suggested we rephrased to “mitochondria in MTCH2 KO cells are sensitized to combined ____GPATi/CHX treatment____.” (page 16, line 4).____ In addition, we performed the nocodazol experiment suggested by the reviewer and included the results in the MS (Rev Supp Fig. 4G-I). The results suggest that MTCH2 KO/GPATi generate clumping and collapse of the mitochondrial network that can be relaxed by blocking microtubule polymerization. Importantly even though the network is still highly fragmented, mitochondrial morphology has changed from large rounded and fragmented to short and tubulated mitochondria, suggesting that MTCH2 KO/GPATi does not impair other mitochondrial architectural changes induced by CHX.

    The fact that FSG67 kills starved cells in the absence of MTCH2 does not mean LPA is not required for starvation induced fusion as concluded by the authors (p.12, first paragraph).

    __R: Thank you and we corrected this part of the MS ____(page 14, line 19)____. __

    __ __ Reviewer #3

    Major comments:

    As FSG67, an inhibitor of glycerol-phosphate acyl transferase (GPAT) for LPA synthesis, blocks GPAT1/2 in the OMM and GPAT3/4 in the ER, it remains possible that OMM-anchored MFN2 cooperates with GPAT1/2 for concentrating LPA at the mitochondrial fusion site. Thus, the authors should test if loss of GPAT3/4, but not GPAT1/2, suppresses mitochondrial elongation in MTCH2 KO cells overexpressing MFN2.

    R: We appreciate the reviewer’s comment and value this observation. We now included in the revised MS two different sets of experiments: silencing ER resident GPATs in MTCH2 KO MEFs, and enforcing mitochondrial fusion either by MFN2 overexpression (Rev Fig. 3L-N) or by HBSS/CHX stress (Rev Fig. 4I-K). We found that ER GPATs do have a contribution to MTCH2-independent mitochondrial fusion enforced by MFN2 overexpression, and to HBSS-induced mitochondrial fusion. These new results suggest that in the absence of MTCH2, LPA synthetized at the ER, is utilized for mitochondrial fusion. Unfortunately, we were unsuccessful in silencing GPAT1 (we tried two different sets of siRNAs, each composed of four different oligos).

    It seems conceivable that FSG67 treatment causes a decrease in the protein levels of MFN2 and/or MTCH2, thereby leading to mitochondrial fragmentation. The authors should clarify this point by western blotting.

    R: We thank the reviewer for this insightful comment. We performed these experiments and the results appear in the MS (Rev Supp Fig. 3A). We analyzed by Western blot the effect of FSG67 (named GPATi in the revised MS) on the expression levels of MTCH2, MFN2, fusion/fission proteins, GPATs, and a few other proteins. Interestingly, GPATi actually resulted in a small increase in MFN2 expression levels but had no effect on MTCH2 expression levels. Moreover, thanks to the reviewer’s suggestion, we also revealed that in non-treated and GPATi-treated MFN2 KO cells, MTCH2 expression levels were increased and GPATs3/4 expression levels were largely decreased (Rev Fig. 3B, C). These results suggest that: 1) Expression of MFN2 is important for the stabilization of GPAT3 and 4 (perhaps they form a functional complex together); 2) the expression levels of endogenous MTCH2 are possibly elevated to compensate for the decreased biosynthesis of LPA and increased demand of LPA funneling for mitochondrial fusion. This hypothesis was added to the discussion.

    Minor comments:

    It would be interesting to investigate whether an ER-anchored MFN2 variant defective in GTP hydrolysis can restore mitochondrial elongation in MTCH2 KO cells.

    R: We thank the reviewer for this suggestion. We generated a MFN2-IYYFT K109A mutant, but unfortunately it was expressed at very low levels, and in the expressed cells it elicited mitochondrial aggregation.

    The authors should add single-color fluorescent images into Figs. 1A, 1C, 1F, S1A, 2H, S2A, S2C, 3C, 3F, S3F, and S3H.

    __R: We thank the reviewer and added single-color fluorescent images into all the Figures requested.

    __

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Goldman et al. report the role of mitochondrial carrier homolog 2 (MTCH2), a mammalian atypical transporter, in regulating mitochondrial shape. Although numerous studies have previously suggested that MTCH2 localizes to the outer membrane of mitochondria (OMM) and acts in a myriad of processes including apoptosis, energy production, mitochondrial dynamics, lipid metabolism, and calcium signaling, its primary function remains uncertain. Using extensive fluorescence imaging, the authors reveal a functional relationship between MTCH2 and MFN1/2, large GTPases regulating mitochondrial fusion. Overexpression of MTCH2 restored mitochondrial elongation in MFN2 KO cells, but not MFN1 KO or MFN1/2 DKO cells. Overexpression of MFN2, but not MFN1, recovered mitochondrial elongation in MTCH2 KO cells. These results suggest that MTCH2 and MFN1 cooperatively act in mitochondrial elongation, and that MFN2 promotes mitochondrial fusion independently of MTCH2 and MFN1. Strikingly, ER-anchored MFN2 can rescue mitochondrial shaping defects in MTCH2 KO cells. The authors also investigated the role of lysophosphatidic acid (LPA), a mitochondrial fusion-promoting lipid, in MTCH2- and MFN2-mediated processes, and found that inhibition of LPA synthesis led to suppression of mitochondrial elongation in MTCH2 KO cells overexpressing MFN2, but not MFN2 KO cells overexpressing MTCH2. Collectively, they propose that MFN1 and MFN2 mediates mitochondrial fusion via two distinct mechanisms: one in the OMM depending on MTCH2 and MFN1, and the other in the ER depending on MFN2 and LPA synthesis.

    Major comments:

    1. As FSG67, an inhibitor of glycerol-phosphate acyl transferase (GPAT) for LPA synthesis, blocks GPAT1/2 in the OMM and GPAT3/4 in the ER, it remains possible that OMM-anchored MFN2 cooperates with GPAT1/2 for concentrating LPA at the mitochondrial fusion site. Thus, the authors should test if loss of GPAT3/4, but not GPAT1/2, suppresses mitochondrial elongation in MTCH2 KO cells overexpressing MFN2.
    2. It seems conceivable that FSG67 treatment causes a decrease in the protein levels of MFN2 and/or MTCH2, thereby leading to mitochondrial fragmentation. The authors should clarify this point by western blotting.

    Minor comments:

    1. It would be interesting to investigate whether an ER-anchored MFN2 variant defective in GTP hydrolysis can restore mitochondrial elongation in MTCH2 KO cells.
    2. The authors should add single-color fluorescent images into Figs. 1A, 1C, 1F, S1A, 2H, S2A, S2C, 3C, 3F, S3F, and S3H.

    Significance

    General assessment:

    The findings in this study are potentially interesting and could provide new insights into the molecular mechanisms of mitochondrial fusion in mammals. The fluorescence imaging data are of high quality with quantification and statistical evaluation, mostly supporting the conclusion. There are, however, some missing points regarding the relationships among MTCH2, MFN1, MFN2, and GPAT1/2/3/4 in more detail. For example, does ER-anchored MFN2 interact with GPAT3/4? Does MTCH2 interact with MFN1 to promote mitochondrial elongation? Is LPA required for MFN1-mediated mitochondrial fusion? Nevertheless, this study would significantly be strengthened if the authors clarify the major and minor comments.

    Advance:

    This study raises the possibilities that ER-anchored MFN2 may act in transport of LPA from the ER to mitochondria in cooperation with GPAT3/4, and that MTCH2 may promote MFN1-mediated mitochondrial fusion independently of LPA.

    Audience:

    Given the findings that ER-anchored MFN2 and LPS synthesis cooperatively acts in promoting mitochondrial fusion, it will attract a broad range of researchers who study mitochondria, ER, membrane fusion, lipids, and interorganellar communication.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Goldman et al examine the role of the mitochondrial protein MTCH2 in mitochondrial fusion. Using a combination of overexpression of MTCH2, MFN1, and MFN2 in respective knockout cells, pharmacological inhibition of GPATs, and stress-induced mitochondrial hyperfusion, the authors explore the relationship between MTCH2, the mitofusins, and LPA levels. These experiments are particularly interesting in light of recent work by Labbe et al which propose that MTCH2 coordinates with LPA to promote mitochondrial fusion. The authors find a key difference from the work of Labbe et al, presenting data suggesting that the role of MTCH2 in starvation-induced mitochondrial hyperfusion varies depending on cell line. They also make the interesting observation that overexpression of MTCH2 is able to bypass GPATi treatment and promote mitochondrial fusion. The authors instead utilize differentially targeted MFN2 to argue that ER-localized MFN2 promotes mitochondrial fusion cooperatively with LPA synthesis. Based on these and other results, they conclude that MTCH2 works with MFN1 to stimulate fusion in a pathway parallel to MFN2 and LPA.

    While the assays are mostly well performed and the findings will be of interest to those in the mitochondrial dynamics field, many of the conclusions drawn by the authors are not supported by the experiments shown. This in turn causes the the text to suffer from a lack of clarity and made the logic of the manuscript hard to follow. Additional controls are also needed to draw robust conclusions from MFN2 targeting and MFN1 overexpression experiments. Finally, the rigor of quantification should be clarified and expanded to all assays in the manuscript.

    Specific points

    1. The authors should comment on recently published work in Science that MTCH1 and likely MTCH2 are outer membrane insertases. The data in the manuscript seem consistent with the model that an undetermined protein may be poorly inserted in a MTCH2 knockout leading to reduced fusion activity mediated by MFN1. Could this explain how MTCH2 overexpression selectively restores fusion to MFN2 KO cells?
    2. The authors make the claim that "MFN2 and MTCH2 compensate for each other's absence" though this is not supported by their data. For example, MFN2 expression is not affected in an MTCH2 KO but cannot compensate to promote fusion. Rather, the authors find that MTCH2 and MFN2 are capable of promoting fusion when overexpressed in the absence of the other.
    3. The authors rely heavily on claims that tagged MFN1 and MFN2 are fully functional, but is it possible that MFN1-GFP is only partially functional? Does untagged MFN1 overexpression cause mitochondrial fusion in a MTCH2 KO? The quantification of this is done only with aspect ratio, and not by categorization of mitochondrial morphology (Fig. S1). The authors should present both analyses in this and all other experiments in the manuscript, particularly since aspect ratio is only performed on 15 cells per condition and not on experimental replicates. The authors should clarify if sample identity was blinded prior to analysis.
    4. The constructs that form the basis of conclusions of ER versus mitochondrial-targeted MFN2 require additional controls to support robust conclusions. The immunofluorescence data suggests that MFN2-ACTA to some degree targets outside of mitochondria (Fig. 2A), and also that MFN2-YIFFT seems to localize to some degree to mitochondria (Fig. 2A). The YIFFT construct may also localize more prevalently to mitochondria in the presence of ACTA (Fig. S2A). Mistargeting would make interpretation of these experiments not possible, as these constructs must exclusively localize to their intended organelle to make strong conclusions. Triple labeling with ER and mitochondrial markers would be helpful, as well as western blots to confirm consistent expression levels and protein stability of each construct. The ACTA MFN2 also appears to promote fusion in a MTCH2 KO. How is this reconciled with the conclusion that ER-targeting of MFN2 is required?
    5. The authors conclude that "MFN2-mediated fusion requires LPA synthesis", but what is shown instead is that GPATi is epistatic to fusion caused by overexpression of MFN2. The authors should be careful about drawing strong conclusions from their overexpression studies. While MTCH2 overexpression causes hyperfusion in the presence of GPATi, this does not mean that LPA doesn't promote MTCH2-dependent fusion, merely that GPATi does not block hyperfusion caused by MTCH2 overexpression.
    6. The collapse of the mitochondrial network in MTCH2 KO cells treated with cycloheximide + GPATi does not indicate the cells "require newly-synthesized LPA to respond to SIMH". Instead, it suggests that mitochondria in MTCH2 KO cells are sensitized to combined GPATi/cycloheximide treatment. Could this collapse be fusion-independent? If mitochondria are treated with nocodazole to relax the mitochondrial network (as in Smirnova et al, MBoC, 2001 or Yang et al, Nat Comm, 2022), does the mitochondrial network appear hyperfused?
    7. The fact that FSG67 kills starved cells in the absence of MTCH2 does not mean LPA is not required for starvation induced fusion as concluded by the authors (p.12, first paragraph).

    Significance

    see above

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, authors investigate the role of MTCH2 in mitochondrial morphology, in several conditions. The authors showed compensatory effect of MFN2 and MTCH2 on stress induced mitochondria hyperfusion (SIMH) in HBSS or CHX treated condition. Since mitochondria hyperfusion upon CHX treatment is impaired in MTCH2 KO cells treated with a GPAT inhibitor, but not in MFN2 KO cells, authors suggest two modes of SIMH, one MTCH2 dependent, the second MFN2/LPA dependent.

    This effect seems to be phenocopied in unstressed condition using overexpression system. Mitochondrial fragmentation in MFN2 KO cells can be recovered by MTCH2 overexpression, and vice versa. The fragmentation of mitochondria in MTCH2 KO MEF is reversed also by an ER-targeted MFN2, suggesting the importance of MFN2 ER localization. The authors also point out that MTCH2 KO have increased mitochondria-ER contacts.

    Major comments

    1. Each of the finding is interesting, but the results are not well discussed and logical links leading to the key conclusions are sometimes missing.

    1-1) A previous report (Bahat et al., 2018) and this manuscript show that MFN2 OE can restore mitochondrial elongation in MTCH2 KO cells and MTCH2 OE can do the same in MFN2 KO cells. Based on these data, authors conclude that "MFN2 and MTCH2 compensate for each other's absence", but the compensation works only when they are overexpressed. On the other hand, upon HBSS or CHX treatment, MFN2 KO or MTCH2 KO cells have elongated/hyperfused mitochondria, but this is not observed in double deficient cells. In this case, MFN2 and MTCH2 show compensatory effects on mitochondria elongation. The authors believe the two conditions, unstressed&OE and stressed conditions, activating same molecular machineries, but it is not fully supported by the data. For example, in unstressed condition, MTCH2 OE can recover MFN2 KO but not of MFN1 KO, suggesting MTCH2-dependent mitochondria fusion requires MFN1, but it is not tested for stressed condition. Furthermore, even if the machineries are common among stressed and unstressed conditions, authors should discuss why the endogenous MFN2/MTCH2 expression is not enough to activate mitochondria fusion in MTCH2/MFN2 KO cells, respectively, and how the compensatory effects are activated upon HBSS or CHX treatment.

    1-2) Authors found out that ER-targeted MFN2 can rescue the mitochondria fragmentation in MTCH2 KO MEF cells, but mitochondria-targeted MFN2 has a lower effect than Wt MFN2. (Fig 2A&B). This finding suggests that MTCH2 loss might impair MFN2 localization at the ER. The authors should investigate endogenous MFN2 localization in MTCH2 KO MEFs.

    1-3) The analysis to test whether recovery of mitochondrial morphology by ER-targeted MFN2 in MTCH2 KO depends on LPA synthesis or not is missing (Fig 3G). Authors should examine whether mitochondrial elongation induced by ER-localized MFN2 in MTCH2 KO cells is impaired by the GPAT inhibitor.

    1-4) In the discussion section, authors suggest that mitochondrial LPA would be a crucial factor for MFN2 dependent mitochondrial fusion. To test this hypothesis, authors should overexpress mitochondrial GPAT and evaluate its effect on mitochondrial morphology.

    1-5) In the discussion section, authors indicated that ER-targeted MFN2 could recover mito-ER contacts leading to LPA flux from ER to mitochondria and mitochondria elongation in MTCH2 KO. However, MTCH2 KO itself already have more mito-ER contacts (Fig 2D-H), and an artificial linker fails to recover mitochondria fragmentation in MTCH2 KO cells (Fig S2C, D). Thus, increased number of contacts appears not sufficient to recover the phenotype. The authors should consider this point in the discussion.

    1. Certain methods are not appropriate to support the stated conclusions.

    2-1) Authors assess "mitochondria fusion" by evaluating mitochondrial morphology. The authors also describe mitochondrial clumping as a fusion-impaired phonotype (Fig 4A&B). Mitochondrial fusion should be evaluated using a PEG assay or a mtPA-GFP analysis.

    2-2) In figure 2D-G, authors show that MTCH2 KO cells have more and longer mitochondria-ER contacts. The correct experiment is not to compare these cells to WT, but to KO reconstituted with MTCH2.

    2-3) Since staining of MitoTracker depends on mitochondrial membrane potential, mitochondria with low potential would be invisible and excluded from the analysis. Authors should investigate mitochondrial morphology by immunostaining also in Fig 4D, S4A, D, and K.

    1. Other points

    3-1) There are some discrepancies with the previous Labbé et al article.: Labbé et al. suggest that MTCH2 activity on mitochondria (from HCT116 cells) fusion is dependent on LPA, based on in vitro fusion assay. On the other hand, this manuscript shows that inhibition of LPA synthesis could not block MTCH2-induced mitochondria elongation in WT MEF and HEK293T cells. MTCH2 KO HCT116 cells are resistant to HBSS- but sensitive to CHX-induced mitochondria elongation. In this manuscript, MTCH2 KO MEF cells are sensitive to both stimuli, and only when MTCH2- and MFN2-deficient MEF cells are resistant to both stimuli. These discrepancies would be caused by difference of assay system or cell lines, but it is not clearly addressed.

    Minor comments

    1. Since authors use FSG67, an inhibitor against GPAT1, 2 and 3, knocking down of each of GPATs will improve the significance of this work.
    2. Recently, a paper about MTCH2 is published (Guna et al., Science), which shows its insertase activity on tail anchored proteins. Authors should include this point of view in the discussion.

    Significance

    Mitochondrial carrier homologue 2 (MTCH2/MIMP/SLC25A50) was found as a mitochondrial solute carrier family member but the substrates are unknown. MTCH2 has roles on apoptosis with Bid (Zaltsman et al., 2010, the authors' group), lipid homeostasis (Rottiers et al., 2017), and mitochondrial morphology (Bahat et al., 2018, the authors' group, and Labbé et al., 2021).

    Stress induced mitochondria hyperfusion (SIMH) was reported in 2009 by Tondera et al.. Under stress condition, such as UV-C, cycloheximide (CHX), or actinomycin D treatment, hyperfused mitochondria were observed and the event was named as SIMH. They also showed that SIMH is dependent on L-Opa1, MFN1 and SLP-2, which is later found as Opa1 regulator, but not on MFN2, BAX/BAK.

    In this manuscript, authors show compensatory effect of MFN2 and MTCH2 on SIMH in HBSS or CHX treated condition. This compensatory effect seems to be reproduced in unstressed condition: mitochondrial fragmentation in MFN2 KO cells can be recovered with MTCH2 overexpression, and vice versa. Authors indicate that LPA synthesis and its mitochondrial localization would be crucial for MFN2 dependent fusion. The compensatory effect of MFN2 and MTCH2 is potentially interesting for a large audience in multiple cell biology fields (mitochondrial biology, ER-mitochondria contact sites, lipid biology).

    Expertise: Mitochondria; fusion/fission; ER-mitochondria contact sites

  6. Version published to 10.1101/2022.10.04.510812v1 on bioRxiv