Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

  1. Yueyang Wang
  2. Lee D Troughton
  3. Fan Xu
  4. Aritra Chatterjee
  5. Chang Ding
  6. Han Zhao
  7. Laura Pulido Cifuentes
  8. Ryan B Wagner
  9. Tianqi Wang
  10. Shelly Tan
  11. Jingjuan Chen
  12. Linlin Li
  13. David Umulis
  14. Shihuan Kuang
  15. Daniel M Suter
  16. Chongli Yuan
  17. Deva Chan
  18. Fang Huang
  19. Patrick W Oakes
  20. Qing Deng

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Abstract

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2 -/- cells maintain a circular shape. Increased cytosolic Ca 2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca 2+ activates Ca 2+ /calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

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  1. Version published to 10.7554/elife.88828 on eLife
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    Reply to the reviewers

    We thank the reviewers for their enthusiasm for our work and constructive feedback.

    Below please find our point-by-point response to the comments:

    Reviewer #1 ** -Key conclusions that were less convincing: -RhoA and NMII are in the title as mechanistic downstream regulators of CaM, but the results in Fig 8 call into question the role of RhoA. Why does RhoA activation not influence cell size and circularity? Can you overexpress MRLC-GFP and inhibit Rho and restore the wt phenotype? The role of NMII is also not clear - why does overexpressing MLCK-CA not have a phenotype but overexpressing MLCK downstream target MRLC show the phenotype? Are there any alternative pathways to regulate MRLC? It's not being discussed or described in 6D's schematic.

    Response: Rho activation usually leads to the formation of more stress fibers and therefore does not lead to decreased cell size and increased circularity observed in MFN2 KO. The phenotype is restored by either ROCK or MLCK knockdown. We have discussed in the main text that the formation of PAB requires both RhoA and NMII activation under restricted spatiotemporal control.

    MRLC has three major regulators (Ikebe & Hartshorne, 1985; Isotani et al., 2004). As we discussed, MLCK and ROCK phosphorylate MRLC at either Ser19 or Ser19 and Thr18. MRLC is dephosphorylated and inactivated by its phosphatase MLCP. We tried to knock down MLCP in wt MEF cells but failed to see any cell morphology changes (data not shown).

    We were also surprised to see MRLC-GFP overexpression with Rho Activator can phenocopy PAB, but “MLCK-CA + Rho Activator” failed to. We believe it is because MLCK-CA constitutively over-activates a broad range of downstream effectors while overexpressing MRLC mimics endogenous activation or NMII alone. Also, only a proportion of cells acquired PAB structure under Rho Activator and MRLC overexpression, which indicates PAB formation also requires specific spatiotemporal controls.

    *Rewrite for clarity -The role of ER/mito contacts in the system was unclear (since ER/mito contacts were not observed nor evaluated directly). *

    __Response: __We have included additional data to measure ER/mito contacts in MEFs. Our result is consistent with numerous previous reports that MFN2 regulates ER/mito contacts. The data is now included in Fig. S3.

    -What role does focal adhesions have on PAB formation or any part of the model - There were results showing larger focal adhesions in the MFN2-/- cells, but not sure how this fits in with the bigger picture of contractility and PABs, and focal adhesions were not in the model in Figure 5.*

    __Response: __Focal adhesion and actomyosin are tightly coupled, and our work focuses on the actin network. Our model did not include FAs since FA is not a significant focus in this study.

    -Whether regulating calcium impacts PAB formation*

    __Response: __Calcium likely regulates PAB formation. We have shown PAB cell percentage decreases in mfn2-/- with ER-mito tethering contrast in Fig. S3.

    * *-The role of PABs in migration is also unclear - can you affect PAB formation or get rid of PABs and quantify cell migration?

    __Response: __Our data suggest that PAB formation and cell migration are inversely correlated. Since PAB results from a contractile actin band on the cell periphery, its role in defective cell spreading and migration is expected. We demonstrated that MLCK and ROCK knockdown reduced PABs and rescued cell spreading.

    -It was hard to understand the correlation between the membrane tension of MFN2-/- cells and their ability to spread on softer substrates. How does this result fit in with the overarching model?

    __Response: __Reduced membrane tension is presumably associated with decreased cell spreading. Softer substrates attenuate the mechanical force on focal adhesion proteins and the actin cytoskeleton (Burridge & Chrzanowska-Wodnicka, 2003; Pelham & Wang, 1997; Wong et al., 2015), which is required for focal adhesion maturation. As a result, softer substrates can reduce the over-contraction in the MFN2 KO cells. The results support the model that MFN2 KO cells have enhanced cell contraction on the substrates dependent on substrate interaction and force transduction on focal adhesions. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    __Response: __We have removed the MFN2-related disease from the introduction to focus the paper more on cell biology in vitro.

    *-There were a number of findings that did not seem to fit in with the paper, or they were included, but were not robustly described nor quantified. As an example, Paxillin-positive focal adhesions were evaluated in MFN2-/- and with various pharmacological approaches, but there is no quantification with respect to size, number, or distribution of focal adhesions, despite language in the main text that there are differences between conditions. *

    __Response: __We have quantified the focal adhesions in the KO cells, and the data is now included in Fig. 5. We used the actin distribution to quantify the “PAB”; therefore, FAs are not a significant focus of this study.

    *Also, the model was presented in figure 5, and then there were several pieces of data presented afterward, some that are included in the model (myosin regulation), and some that are not in the model (membrane tension, TFM, substrate stiffness, etc). * __Response: __Membrane tension and substrate stiffness dependence are physical properties of the cell. The model focuses on the molecular mechanism that leads to PAB formation.

    The stiffness/tension figure was not clear to me, and it was difficult to make sense of the data since one would predict that an increase in actomyosin contractility at the cortex would lead to higher membrane tension, not lower, and then how membrane tension relates to spreading on soft matrices is also unclear.

    __Response: __The result was surprising to us initially. However, the MFN2 KO cells have increased actomyosin contractility only at the cell-substrate interface but not throughout the entire cell cortex. A less spread cell would have a more relaxed membrane and display a lower membrane tension, consistent with our observation. Softer matrices reduce cell contractility at the cell-substrate interface, which allows MFN2 KO cells to relax and spread better. We have emphasized in the discussion of our manuscript that MFN2 KO cells have an increase in actomyosin contractility only at the cell-substrate interface.

    The manuscript seems like an amalgamation of different pieces of data that do not necessarily fit together into a cohesive story, so the authors are encouraged to either remove these data, or shore them up and weave them into the narrative.

    __Response: __We respectfully disagree with the reviewer since the cell morphology, actin structure, substrate interaction, and cell mechanics are tightly correlative and provide a complete picture of the role of MFN2 in regulating cell behavior.

    Request additional experiments

    1. -The imaging used for a lot of the quantification (migration, circularity) is difficult to resolve. The cells often look like they are not imaged in the correct imaging plane. It would be helpful to have better representative images such that it is clear how the cells were tracked and how cell periphery regions of interest were manually drawn. Focal adhesions should also be shown without thresholding.*

    __Response: __We used a TIRF microscope and imaged a Z stack. Imaris was used to combine the Z-stack images. The images in the manuscripts are now of the lowest stack with background subtraction.

    2. -For most of the quantification, it appears that the experiment was only performed once and that a handful of cells were quantified. The figure legend indicates the number of cells (often reported as >12 cells or >25 cells), but the methods indicate that high content imaging was performed, and so the interpretation is that these experiments were only performed once. Biological replicates are required. If the data do represent at least 3 biological replicates, then more cell quantification is required (12 or 25 cells in total would mean quantifying a small number of cells per replicate).

    Response: We quantified more cells and indicated the number of cells quantified in the figure legends. The experiments are with three biological replicates.

    1. -Mitochondrial morphology and quantification should be performed in the MFN2 knockdown and rescue lines.*

    __Response: __Mitochondrial morphology is well characterized in the Mfn2 KO and rescue MEF cells (Chen et al., 2003; Naon et al., 2016; Samanas et al., 2020). We observed a similar phenotype using mito-RFP to label mitochondrial structure (Fig. S1).

    4.-Many of the comparisons throughout the figures is between MFN2 knockdown and MFN2 knockdown plus rescue or genetic/pharmacological approaches, but a comparison that is rarely made is between wildtype and experimental. These comparisons could be useful in comparing partial rescues and potential redundancies with the other mitofusin.

    Response: We have included the WT in our assays (Fig. 2-6). We also confirmed that MFN1 could not rescue the MFN2 defects (Fig. 2). We observed partial and complete rescue in different assays. It would be difficult to conclude whether the phenotype is due to the redundancies with the other mitofusin because not all cells are rescued at the endogenous level.

    1. -For the mito/ER tethering experiments, it is important to show that ER/mito contacts are formed and not formed in the various conditions with imaging approaches.*

    __Response: __We adopted a previously established method to quantify ER-mitochondria contacts with the probe SPLICSL (Cieri et al., 2017; Vallese et al., 2020). Our results align with previous reports that Mfn2-null MEFs displayed significantly decreased ER-mitochondria contacts. MFN2 re-expression or ER-mitochondria tethering structure restored the contacts. (Fig. S3).

    1. -For some of the pharmacological perturbations, it would be helpful to show that the perturbation actually led to the expected phenotype - as an example, in cells treated with different concentrations of A23187, what are the intracellular calcium levels and how do these treatments influence PAB formation? This aspect should be generally applied across the study - when a modification is made, that particular phenotype should first be evaluated, before dissecting how the perturbation affects downstream phenotypes.*

    __Response: __We selected a collection of well-characterized inhibitors broadly used in the literature for pharmacological perturbations. For example, numerous studies used A23187 treatment to raise intracellular calcium to examine related actin cytoskeleton changes (Carson et al., 1994; Goldfine et al., 1981; Shao et al., 2015). We titrated the drugs in WT in preliminary experiments and observed similar phenotypes. (data not shown). We then use the same concentration to treat the MFN2 KO cells. Overall, we use pharmacological perturbations as supporting evidence. We use genetics (knockdown or overexpression) to validate our results.

    7. -In Figures 4 and 5, the thresholding approaches in the images make the focal adhesions difficult to resolve, and therefore it is difficult to determine the size. As described above, these metrics should be defined and quantified.

    __Response: __We used a TIRF microscope and imaged a Z stack. Imaris was used to combine the Z-stack images. The images in the manuscripts are now of the lowest stack with background subtraction.

    8. -What is a PAB? How is it defined? What metrics make a structure a PAB versus regular cortical actin - are there quantifiable metrics? In figure 8, there are some structures that are labelled as a PAB, but some aren't (as an example, the left panel in 8b is a PAB, but the right panel in 8A is not, but they look the same), so a PAB should be defined with quantifiable measures, and then applied to the entire study.

    __Response: __We developed an algorism to quantify PAB cells. We first used the ImageJ plugin FiloQuant (Jacquemet et al., 2019) to identify the cell border and cytoskeleton, then used our custom algorism to quantify the percentage of actin in the cell border area. The cellular circularity is also calculated at the same time. If the cell contains more than 50% actin in the cell border area, and the circularity is higher than 0.6, we then count it as a “PAB” cell (Fig. S2).

    -As described above, why does RhoA activation not influence cell size and circularity? Can you overexpress MRLC-GFP and inhibit Rho and restore the wildtype phenotype?

    __Response: __Rho activation usually leads to the formation of more stress fibers and therefore does not lead to decreased cell size and increased circularity observed in MFN2 KO.

    We are sorry that we don’t understand the rationale of this experiment proposed by the reviewer. ROCK inhibition restored the wildtype phenotype in MFN2 KO cells (Fig.7). Figure 8 is to create the MFN2 KO phenotype in WT cells, which requires both Rho and MRLC overactivation.

    10 Are the data and the methods presented in such a way that they can be reproduced? -We appreciate that the authors quantified many parameters, although some quantifications were missing. There are some missing methods - how was directionality quantified, was migration quantified by selecting the approximate center of cells using MTrackJ or were centroids quantified by outlining cells, for instance. Also, given that some of the phenotypes were somewhat arbitrarily assigned (ie. what constitutes a PAB?), it may be difficult to reproduce these approaches and interpret data appropriately.*

    __Response: __We have clarified directionality quantification methods and other details. We used MTrackJ to track cell migration. And as we mentioned above, we came up with a customized algorithm to quantify PAB cells, which shows the critical effectors in a more quantifiable way.

    1. Are the experiments adequately replicated and statistical analysis adequate? -Unfortunately, while the approximate number of cells was reported, the number of biological replicates were not reported, and therefore, the experimental information and statistical analyses are not adequate.*

    __Response: __We have quantified more cells and indicated the number of repeats and cells quantified in the figure legend. Minor comments: Specific experimental issues that are easily addressable.

      • for some of the graphs - mostly about calcium levels - fold change is shown, but raw values should also be included to determine whether the basal levels of calcium are different across the conditions.*

    __Response: __Delta F/F0 is the standard method to normalize dye loading in cells for calcium concentration measurements (Kijlstra et al., 2015; Zhou et al., 2021).

    13. - scale bars in every panel should also help make the points clearer.

    __Response: __We have added scale bars in all panels.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    1. Fig. 1A: The Mfn1 Western Blot is not of publication quality. Moreover, quantitation is necessary.*

    __Response: __We performed additional western blots, changed the representative images, and quantified the level of knockdown and overexpression (Fig.2 and 7). We did not quantify the WB in Fig.1A since it was to confirm that the Mfn1-/- or Mfn2-/- were knock-out cell lines.

    2. Fig. 1B (as well as Fig. 2G and others): the date do not reflect cellular size but instead spread cellular area.

    __Response: __We thank the reviewer for this suggestion. We have changed all similar descriptions to “Spread Area” in the main text and figures.

    3. Fig. 1C, D: Mfn1-null MEFs appear to be more spindle-shaped than wt cells, yet their circularity tends to be elevated. Do the authors have an explanation?

    __Response: __The circularity of Mfn1-/- MEFs has a slight increase but is not significant compared to the wt cells. As we observed, Mfn1-/- MEFs have fewer protrusions than wt, which may contribute to the slight increase in its circularity (Fig. 5C). However, this is not the focus of this study.

    1. Fig. 2A: The Mfn1 levels in Mfn2-/- + Mfn2 are lower than Mfn2-/-? Does this imply a crosstalk between Mfn1 and Mfn2 expression.*

    __Response: __We agree with the reviewer that a compensatory change in MFN1 expression might happen in Mfn2-/- + MFN2 MEFs. Previous research also indicated crosstalk between MFN1 and MFN2 expression (Sidarala et al., 2022).

    1. Fig. 2H: The authors should provide co-staining of mitochondria and Mfn2 as well.*

    __Response: __Co-staining of mitochondria and MFN2 in Mfn2-/- MEFs or rescue lines has been done in numerous previous studies (Chen et al., 2003; Naon et al., 2016; Samanas et al., 2020). In this work, we transfected our cells with mito-RFP and showed mitochondria changes in Mfn2-/- and rescue MEF cells (Fig. S1G).

    6. Fig. 4D-E: Western blots are not of publication quality. Looking at the blots provided in Fig. 4D, the reviewer is not convinced with the quantitative data shown in Fig. 4E. For instance, the intensity of pCaMKII band for "vec" does not look 3x higher than that of "+MFN2", whereas that of "+MFN2" looks much higher than that of WT.

    __Response: __We have performed additional western blots and changed the representative images.

    1. Fig. 5C: The authors should stain for vinculin, which are present in mature FAs only, rather than paxillin which are present in all FAs. This would strengthen the authors' conclusions. Also, FA size should be quantified.*

    __Response: __We have quantified FA size in Figure 5. The maturity of FAs is not a major focus of this study. It is likely that most FAs here are mature since they are connected with stress fibers.

    1. Fig. 6C - Why does the background have a grid and appear grey in color? Also, the cell interior appears in different colors in the different images. The authors should take a z-stack of images and provide the raw image files.*

    __Response: __We used a TIRF microscope and imaged a Z stack. Imaris was used to combine the Z-stack images. The images in the manuscripts are now of the lowest stack with background subtraction.

    9. Fig 7C: The MLCII Western blot is not of publication quality, and may affect the quantification provided in Fig. 7D.

    __Response: __We have performed additional western blots and changed the representative images.

    1. Fig 8: Do cell treatments with Rho Activator and MLCK-CA also impair migration velocity similar to Mfn2-null cells?*

    __Response: __Our data indicated that Rho Activator and MRLC induced the “PAB” structure seen in MFN2 KO cells. It is likely that cell migration is impaired here. Spatiotemporal regulation of Rho Activation is important to cell migration, it is known that Rho overactivation can significantly inhibit cell migration (Nobes & Hall, 1999). Showing Rho Activator and MLCK-CA will reduce cell migration will not add new knowledge to the cell migration field. However not all cell migration defects are associated with the PAB. We, therefore, focused on PAB quantification in this figure.

    11. Fig. 9A: The authors claim that wt cells have actin bundles that protrude against the membrane while Mfn2-null cells do not. This does not look convincing as the Mfn2-null actin bundle seem to be pushing against the membrane at the bottom of the image. No quantification is provided. It is unclear what conclusion can be drawn from the super-resolution images.

    __Response: __We used super-resolution imaging to demonstrate the details of the peripheral actin band (PAB) structure. We have used two boxes to enlarge the regions where membrane parallel actin structures are predominant. The quantification of PAB is provided in other figures.

    12. Suppl. Fig. 5C: The authors should take images using a confocal microscope for cells with Flipper-TR construct, eliminate the background and the cell center to only consider the cell periphery. Nikon TE2000 does not seem to be a confocal microscope.

    Response: The amount of Flipper-TR that MEF cells can take in was limited. With the current signal-to-noise ratio, complete background elimination is not feasible. A confocal microscope is not necessary for Flipper-TR FLIM imaging (García-Calvo et al., 2022).* ** Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    General Comments (Major)

    1. Data Presentation and analysis: The data analysis would benefit from using a method such as Super-Plots to show the data from separate biological repeats and to use N numbers that represent the number of biological repeats rather than the number of cells analysed. Please see the following reference for a suggestion on how to analyse the data: Lord SJ, Velle KB, Mullins RD, Fritz-Laylin LK. SuperPlots: Communicating reproducibility and variability in cell biology. J Cell Biol. 2020 Jun 1;219(6):e202001064. doi: 10.1083/jcb.202001064. PMID: 32346721; PMCID: PMC7265319.*

    __Response: __We have changed the dot colors to show data from separate biological repeats.

    • Another general comment is that many of the experiments show analysis of very few numbers of cells or maybe only one field of view in a microscopy quantification experiment. This seems unusually low - for example, in Figure 1E only 6 cells have been analysed. It seems like more could have been done and if statistical analysis like we suggest in 1 above is used, this might reveal that some of the differences are less significant than the authors think/report. This is important, as cells are noisy and it is unusual to have such high significance for experiments like cell migration and other parameters unless a lot of measurements are made. In Figure 1G, it appears that only one field of view has been used to quantify the data. We routinely use 3-5 fields of view to get a representative sample of what the cells are doing.*

    __Response: __We have quantified more cells and indicated the number of repeats and cells quantified in the figure legend.

    • Some of the micrographs appear to be missing scale bars- e.g. Fig. 2H, Fig. 8*

    __Response: __We have included scale bars in the lower right corner of all panels.

    1. In the cell tracking experiments, only some of the cells in the images appear to have been tracked. How were the tracked cells chosen? Normally, we would track every cell to avoid bias in selection.*

    __Response: __We tracked all the cells in the view at the beginning of the experiments.

    5. The western blot images do not show the molecular size of the bands. Show ladder position

    __Response: __We have added bands to show molecular weights.

    6. Mostly the graphs show individual data points, which is good, but in some cases only a bar is shown- it would be nice to have individual points overlaid on the bars- e.g. Figure 1I, 4E, 5B, 5E, 7B, 7D

    __Response: __We have updated the graph to show individual points.

    7. Many of the confocal images look very processed- they have no background and have a hazy black halo around the cell. I am not familiar with the type of processing that was done and I worry that the images are only showing a masked and processed version of the actual data. The authors need to explain what processing they have done and probably also to provide the unprocessed images in a supplementary figure or dataset for readers to see. The methods description is inadequate as it only says Image J was used to process the data.

    __Response: __We used a TIRF microscope and imaged a Z stack. Imaris was used to combine the Z-stack images. The images in the manuscripts are now of the lowest stack with background subtraction.

    Individual comments on Figures: Figure 1: See general comments above- consider to use Superplots, more cells and more fields of view in quantifications. Show experimental points in bar graph.*

    __Response: __We have quantified more cells and used super plots to display the data. The number of repeats and cells quantified are indicated in the figure legend.

    Figure 2: In 2E, the colours are very similar for two of the experiments so it is difficult to distinguish them- e.g. the MFN1 vs MFN2 rescue data both appear dark blue. Response: We have changed the color for MFN1 rescue to distinguish the two samples better.

    *In 2H are the magnifications really the same for the WT as the +DOX and -DOX? The cell in the WT looks huge. Is this representative? *Also, the phalloidin stain looks very spotty on the WT. This seems unusual.

    __Response: __The images are of the same scale. The Mfn2-/- MEFs are smaller, and DOX-induced MFN2 expression can only partially rescue the cell size.

    Figure 3: Not many cells were analysed in 3B, especially the zero time point.*

    __Response: __We have quantified more cells.

    Please define +T, we assume it is the tether construct, but it is not defined*

    __Response: __We defined T as a tether in the main text and the figure legend. In 3F, how were the tracked cells chosen?

    __Response: __We tracked all the cells in the view at the beginning of the experiments.

    Figure 4:** 4B: Why have they not tested FK506 and STO609 on the WT cells?*

    __Response: __We focused on understanding the MFN2 KO phenotype. Since neither FK506 nor STO609 altered the MFN2 KO phenotype, we did not include them in the WT group.

    4C: How were the tracked cells chosen?

    __Response: __We tracked all the cells in the view at the start of the experiments.

    4D-E: The blot doesn't look representative of the quantification- were the numbers normalised to vinculin? The difference between WT and vector looks too large to be real if the amounts were normalised to the vinculin, as vinculin is increased in vector. Likewise, the pCAMKII looks to be substantially decreased from the +MFN2, but this is not what the quantification shows.

    __Response: __We have performed additional western blots and changed the representative images.

    Figure 5:* 5B- please clarify which ratio is shown here. I assume it is the ratio of RhoA-GTP vs RhoA between the zero and 4 minute time points.*

    __Response: __Yes, we have clarified this point in the figure legend.

    5C- These images appear to have a mask around the cell. It is hard to tell where the edge of the cell really is- what sort of processing was used? Especially for the paxillin staining, why is there no cytoplasm shown? Is this because the image is in TIRF?

    __Response: __We used a TIRF microscope and imaged a Z stack. Imaris was used to combine the Z-stack images. The images in the manuscripts are now of the lowest stack with background subtraction.

    Figure 6: Figure 6C- the blebbistatin treated cell looks very large- is this representative?

    __Response: __Yes, Blebbistatin-treated cells are larger (Fig. 6A).

    Figure 7: Fig 7C- The lanes for MLCII are all run together- is this from a different gel? Is this quantification accurate?

    __Response: __We have performed additional western blots and changed the representative images.

    Fig. 7F- What is the % level of knockdown achieved?

    __Response: __The level of knockdown is labeled on the figure.

    Figure 8:* Fig 8A,B- does the scale bar represent all of the images in these two panels?*

    __Response: __Yes, the figure legend is updated to clarify this point.

    Fig 8C,D- Superplots would be helpful here.

    __Response: __We have used super plots to display the data.

    Supplementary Data:* The OCR data do not add much and are not discussed much in the manuscript. Perhaps they could be omitted.*

    __Response: __Our OCR data ruled out the possibility of metabolic regulation. Since MFN2 is a mitochondria protein with its typical functions in metabolic pathways, we cannot omit its influence on metabolism here. As we observed, shMLCK enhanced OCR, shROCK reduced OCR, and both knock-down rescued cell morphology and motility. We believe that PAB formation is independent of MFN2’s function in metabolic regulation.

    Figure S3- The figure label doesn't match the manuscript test- was fibrinogen or collagen used?

    __Response: __We tried cover glass alone, collagen, and fibronectin-coated glass. The PAB formation is independent of these extracellular substrates. We did not try fibrinogen because MEF cell is reported to prefer fibronectin (Lehtimäki et al., 2021).

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    Referee #3

    Evidence, reproducibility and clarity

    This study explores the importance of MFN2, a known endoplasmic reticulum-mitochondria linker protein, in cell motility and actin-myosin organization. It is known that the mitofusin proteins MFN1 and MFN2 can tether the mitochondria to the ER and are connected with calcium regulation in muscle and non-muscle cell types. Calcium flux mediated by mitofusins has previously been implicated in apoptosis and ER stress. In this study, the authors show that loss or depletion of MFN2 (but not MFN1) can lead to aberrant calcium increase in the cytoplasm and trigger actin-myosin reorganisation. They show that the actin and myosin changes are linked with activation of RhoA and also that they can be suppressed by inhibiting myosin light chain phosphorylation/ activation. They also show that cells with reduced MFN2 are softer (using atomic force microscopy), which agrees with activation of RhoA and contractility. If cells are plated on softer substrata, it partially compensates for the over-activation of RhoA.

    In many places, the data support the claims made, but in several places the experiments could be made more convincing or be more clearly presented. Some of the experiments appear to have been repeated only 1-2 times and very few cells or fields of view have been analysed.

    General Comments (Major)

    1. Data Presentation and analysis: The data analysis would benefit from using a method such as Super-Plots to show the data from separate biological repeats and to use N numbers that represent the number of biological repeats rather than the number of cells analysed. Please see the following reference for a suggestion on how to analyse the data: Lord SJ, Velle KB, Mullins RD, Fritz-Laylin LK. SuperPlots: Communicating reproducibility and variability in cell biology. J Cell Biol. 2020 Jun 1;219(6):e202001064. doi: 10.1083/jcb.202001064. PMID: 32346721; PMCID: PMC7265319.
    2. Another general comment is that many of the experiments show analysis of very few numbers of cells or maybe only one field of view in a microscopy quantification experiment. This seems unusually low - for example, in Figure 1E only 6 cells have been analysed. It seems like more could have been done and if statistical analysis like we suggest in 1 above is used, this might reveal that some of the differences are less significant than the authors think/report. This is important, as cells are noisy and it is unusual to have such high significance for experiments like cell migration and other parameters unless a lot of measurements are made. In Figure 1G, it appears that only one field of view has been used to quantify the data. We routinely use 3-5 fields of view to get a representative sample of what the cells are doing.
    3. Some of the micrographs appear to be missing scale bars- e.g. Fig. 2H, Fig. 8
    4. In the cell tracking experiments, only some of the cells in the images appear to have been tracked. How were the tracked cells chosen? Normally, we would track every cell to avoid bias in selection.
    5. The western blot images do not show the molecular size of the bands.
    6. Mostly the graphs show individual data points, which is good, but in some cases only a bar is shown- it would be nice to have individual points overlaid on the bars- e.g. Figure 1I, 4E, 5B, 5E, 7B, 7D
    7. Many of the confocal images look very processed- they have no background and have a hazy black halo around the cell. I am not familiar with the type of processing that was done and I worry that the images are only showing a masked and processed version of the actual data. The authors need to explain what processing they have done and probably also to provide the unprocessed images in a supplementary figure or dataset for readers to see. The methods description is inadequate as it only says Image J was used to process the data.

    Individual comments on Figures:

    Figure 1: See general comments above- consider to use Superplots, more cells and more fields of view in quantifications. Show experimental points in bar graph.

    Figure 2: In 2E, the colours are very similar for two of the experiments so it is difficult to distinguish them- e.g. the MFN1 vs MFN2 rescue data both appear dark blue. In 2H are the magnifications really the same for the WT as the +DOX and -DOX? The cell in the WT looks huge. Is this representative? Also, the phalloidin stain looks very spotty on the WT. This seems unusual.

    Figure 3: Not many cells were analysed in 3B, especially the zero time point. Please define +T, we assume it is the tether construct, but it is not defined In 3F, how were the tracked cells chosen?

    Figure 4: 4B: Why have they not tested FK506 and STO609 on the WT cells? 4C: How were the tracked cells chosen? 4D-E: The blot doesn't look representative of the quantification- were the numbers normalised to vinculin? The difference between WT and vector looks too large to be real if the amounts were normalised to the vinculin, as vinculin is increased in vector. Likewise, the pCAMKII looks to be substantially decreased from the +MFN2, but this is not what the quantification shows.

    Figure 5: 5B- please clarify which ratio is shown here. I assume it is the ratio of RhoA-GTP vs RhoA between the zero and 4 minute time points. 5C- These images appear to have a mask around the cell. It is hard to tell where the edge of the cell really is- what sort of processing was used? Especially for the paxillin staining, why is there no cytoplasm shown? Is this because the image is in TIRF?

    Figure 6: Figure 6C- the blebbistatin treated cell looks very large- is this representative?

    Figure 7: Fig 7C- The lanes for MLCII are all run together- is this from a different gel? Is this quantification accurate? Fig. 7F- What is the % level of knockdown achieved?

    Figure 8: Fig 8A,B- does the scale bar represent all of the images in these two panels? Fig 8C,D- Superplots would be helpful here.

    Supplementary Data:

    The OCR data do not add much and are not discussed much in the manuscript. Perhaps they could be omitted. Figure S3- The figure label doesn't match the manuscript test- was fibrinogen or collagen used?

    Significance

    The main novelty here appears to be the connection between excess cytoplasmic calcium, MFN2 loss and RhoA/myosin activation. This is interesting and a useful addition to the literature. It is unclear what the significance is perhaps, as increased cytoplasmic calcium is likely to cause multiple effects in addition to these. So, this effect may be a side-effect of uncoupling the ER and the mitochondria. Nonetheless, it is important to know about this and it will likely inform future studies on the mitofusins.

    This will be of interest to basic researchers studying mitochondria function and the connections between signaling and mitochondria function.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Wang et al. investigate the role of Mitofusin 2 (Mfn2) in MEF morphology and migration. MEFs lacking Mfn2 exhibit a rather circular shape and reduced migratory potential due to increased cytosolic Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II, RhoA and myosin light-chain kinase (MLCK), triggering non-muscle myosin II overactivation and formation of an F-actin ring at the cell periphery. Overall, this is an interesting study. However, additional work is needed for this manuscript to reach publication quality.

    Major Concerns:

    • Several of the Western blots are not of publication quality. This may affect their quantification, and the authors' conclusions.
    • The authors should examine how treatment of wt MEFs with Rho Activator and MLCK-CA affect cell motility (point #10).
    • The reviewer is not convinced that the membrane tension measurements were performed correctly (see point #12 below). The reviewer would expect that the presence of an F-actin ring at the cell periphery would increase the membrane tension, which is opposite to the authors' findings.

    Specific Comments:

    1. Fig. 1A: The Mfn1 Western Blot is not of publication quality. Moreover, quantitation is necessary.
    2. Fig. 1B (as well as Fig. 2G and others): the date do not reflect cellular size but instead spread cellular area.
    3. Fig. 1C, D: Mfn1-null MEFs appear to be more spindle-shaped than wt cells, yet their circularity tends to be elevated. Do the authors have an explanation?
    4. Fig. 2A: The Mfn1 levels in Mfn2-/- + Mfn2 are lower than Mfn2-/-? Does this imply a crosstalk between Mfn1 and Mfn2 expression.
    5. Fig. 2H: The authors should provide co-staining of mitochondria and Mfn2 as well.
    6. Fig. 4D-E: Western blots are not of publication quality. Looking at the blots provided in Fig. 4D, the reviewer is not convinced with the quantitative data shown in Fig. 4E. For instance, the intensity of pCaMKII band for "vec" does not look 3x higher than that of "+MFN2", whereas that of "+MFN2" looks much higher than that of WT.
    7. Fig. 5C: The authors should stain for vinculin, which are present in mature FAs only, rather than paxillin which are present in all FAs. This would strengthen the authors' conclusions. Also, FA size should be quantified.
    8. Fig. 6C - Why does the background have a grid and appear grey in color? Also, the cell interior appears in different colors in the different images. The authors should take a z-stack of images and provide the raw image files.
    9. Fig 7C: The MLCII Western blot is not of publication quality, and may affect the quantification provided in Fig. 7D.
    10. Fig 8: Do cell treatments with Rho Activator and MLCK-CA also impair migration velocity similar to Mfn2-null cells?
    11. Fig. 9A: The authors claim that wt cells have actin bundles that protrude against the membrane while Mfn2-null cells do not. This does not look convincing as the Mfn2-null actin bundle seem to be pushing against the membrane at the bottom of the image. No quantification is provided. It is unclear what conclusion can be drawn from the super-resolution images.
    12. Suppl. Fig. 5C: The authors should take images using a confocal microscope for cells with Flipper-TR construct, eliminate the background and the cell center to only consider the cell periphery. Nikon TE2000 does not seem to be a confocal microscope.

    Significance

    Delineating the role of Mfn2 in cell migration will represent a good, fundamental contribution to the field of cell migration. The reviewer finds this manuscript to be conceptually interesting. Unfortunately, there are technical issues, which need to be fixed so that the readers feel confident about the conclusion of this study.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Wang et al use a combination of cell biological and biochemical approaches to show that Mitofusin2 (MFN2) - a protein typically known to regulate mitochondrial morphology - regulates cell morphology by regulating calcium levels and downstream cell contractility players. They show that cells deficient in MFN2 exhibit increased intracellular calcium levels, and overactivation of myosin II, leading to increased cell contractility. Furthermore, MFN2-deficient cells exhibit an F-actin ring around the cell periphery (which they call PABs). The study takes advantage of both pharmacological and genetic perturbations, as well a variety of assays to support many of their findings; however, it is unclear how these findings are related to each other. Furthermore, MFN2-related disease was raised a few times in the abstract and throughout the manuscript, but it's unclear how the findings in the paper relate to disease states, both in terms of the cell biology, as well as the model that was used (MEFs). This reviewer applauds the authors for exploring MFN2 function outside of its conventional role in mitochondria; but it was difficult to parse through the findings to resolve a mechanistic explanation for how MFN2 affect cell behavior, and what role, if any, PABs have on biological function. While it is important to dissect mitochondrial-independent functions for MFN2, given the whole scale changes in cells in MFN2-deficient cells, and the fact that there is a metabolic phenotype, it is difficult to know how many of the observed phenotypes are downstream of perturbed mitochondrial function versus on cytoskeletal dynamics directly.

    Major comments:

    Are the key conclusions convincing?

    • Key conclusions that were convincing:
      • MFN2-/- cells exhibit decreased cell velocity, decreased cell size, increased cell circularity, and increased intracellular calcium
      • modifying the levels of calcium has an effect on cell circulariy.
      • MFN2-/- cells exhibit increased activation of contractility players
    • Key conclusions that were less convincing:
      • RhoA and NMII are in the title as mechanistic downstream regulators of CaM, but the results in Fig 8 call into question the role of RhoA. Why does RhoA activation not influence cell size and circularity? Can you overexpress MRLC-GFP and inhibit Rho and restore the wt phenotype? The role of NMII is also not clear - why does overexpressing MLCK-CA not have a phenotype but overexpressing MLCK downstream target MRLC show the phenotype? Are there any alternative pathways to regulate MRLC? It's not being discussed or described in 6D's schematic.
      • The role of ER/mito contacts in the system was unclear (since ER/mito contacts were not observed nor evaluated directly).
      • What role does focal adhesions have on PAB formation or any part of the model - There were results showing larger focal adhesions in the MFN2-/- cells, but not sure how this fits in with the bigger picture of contractility and PABs, and focal adhesions were not in the model in Figure 5.
      • Whether regulating calcium impacts PAB formation
      • The role of PABs in migration is also unclear - can you affect PAB formation or get rid of PABs and quantify cell migration?
      • It was hard to understand the correlation between the membrane tension of MFN2-/- cells and its ability to spread on softer substrates. How does this result fit in with the overarching model?

    Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    • There were a number of findings that did not seem to fit in with the paper, or they were included, but were not robustly described nor quantified. As an example, Paxillin-positive focal adhesions were evaluated in MFN2-/- and with various pharmacological approaches, but there is no quantification with respect to size, number, or distribution of focal adhesions, despite language in the main text that there are differences between conditions. Also, the model was presented in figure 5, and then there were several pieces of data presented afterward, some that are included in the model (myosin regulation), and some that are not in the model (membrane tension, TFM, substrate stiffness, etc). The stiffness/tension figure was not clear to me, and it was difficult to make sense of the data since one would predict that an increase in actomyosin contractility at the cortex would lead to higher membrane tension, not lower, and then how membrane tension relates to spreading on soft matrices is also unclear. The manuscript seems like an amalgamation of different pieces of data that do not necessarily fit together into a cohesive story, so the authors are encouraged to either remove these data, or shore them up and weave them into the narrative.

    Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    • The imaging used for a lot of the quantification (migration, circularity) is difficult to resolve. The cells often look like they are not imaged in the correct imaging plane. It would be helpful to have better representative images such that it is clear how the cells were tracked and how cell periphery regions of interest were manually drawn. Focal adhesions should also be shown without thresholding.
    • For most of the quantification, it appears that the experiment was only performed once and that a handful of cells were quantified. The figure legend indicates the number of cells (often reported as >12 cells or >25 cells), but the methods indicate that high content imaging was performed, and so the interpretation is that these experiments were only performed once. Biological replicates are required. If the data do represent at least 3 biological replicates, then more cell quantification is required (12 or 25 cells in total would mean quantifying a small number of cells per replicate).
    • Mitochondrial morphology and quantification should be performed in the MFN2 knockdown and rescue lines.
    • Many of the comparisons throughout the figures is between MFN2 knockdown and MFN2 knockdown plus rescue or genetic/pharmacological approaches, but a comparison that is rarely made is between wildtype and experimental. These comparisons could be useful in comparing partial rescues and potential redundancies with the other mitofusin.
    • For the mito/ER tethering experiments, it is important to show that ER/mito contacts are formed and not formed in the various conditions with imaging approaches
    • For some of the pharmacological perturbations, it would be helpful to show that the perturbation actually led to the expected phenotype - as an example, in cells treated with different concentrations of A23187, what are the intracellular calcium levels and how do these treatments influence PAB formation? This aspect should be generally applied across the study - when a modification is made, that particular phenotype should first be evaluated, before dissecting how the perturbation affects downstream phenotypes.
    • In Figures 4 and 5, the thresholding approaches in the images make the focal adhesions difficult to resolve, and therefore it is difficult to determine the size. As described above, these metrics should be defined and quantified.
    • What is a PAB? How is it defined? What metrics make a structure a PAB versus regular cortical actin - are there quantifiable metrics? In figure 8, there are some structures that are labelled as a PAB, but some aren't (as an example, the left panel in 8b is a PAB, but the right panel in 8A is not, but they look the same), so a PAB should be defined with quantifiable measures, and then applied to the entire study.
    • As described above, why does RhoA activation not influence cell size and circularity? Can you overexpress MRLC-GFP and inhibit Rho and restore the wildtype phenotype?

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    • These suggested experiments described above will take a substantial amount of time and money, as it appears that some experiments were only performed once, and therefore many of these experiments might need to be performed 2-3 more times. Also, experiments showing that addition of drugs lead to expected outcomes prior to analyzing downstream phenotypes will also require a significant amount of time. It is hard to predict how long it will take, but we would guess, 6-8 months?

    Are the data and the methods presented in such a way that they can be reproduced?

    • We appreciate that the authors quantified many parameters, although some quantifications were missing. There are some missing methods - how was directionality quantified, was migration quantified by selecting the approximate center of cells using MTrackJ or were centroids quantified by outlining cells, for instance. Also, given that some of the phenotypes were somewhat arbitrarily assigned (ie. what constitutes a PAB?), it may be difficult to reproduce these approaches and interpret data appropriately.

    Are the experiments adequately replicated and statistical analysis adequate?

    • Unfortunately, while the approximate number of cells was reported, the number of biological replicates were not reported, and therefore, the experimental information and statistical analyses are not adequate.

    Minor comments:

    Specific experimental issues that are easily addressable.

    • for some of the graphs - mostly about calcium levels - fold change is shown, but raw values should also be included to determine whether the basal levels of calcium are different across the conditions.
    • scale bars in every panel should also help make the points clearer.

    Are prior studies referenced appropriately?

    • Yes

    Are the text and figures clear and accurate?

    • Some of the data in the figures were unclear - see above for more info.

    Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    • See above for more info.

    Significance

    Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    • There is a growing field of mitochondrial biology and how it relates to cell migration. This paper examines the function of a key mitochondrial morphology regulator, MFN2, and dissects a role for MFN2 in migration at the level of cytoskeletal regulation. We think that this is interesting, and that it's clear that MFN2 has multiple functions in the cell, but the phenotypes are so pleiotropic that it's difficult to parse out mechanistic understanding. The authors also describe a new actin architecture - a structure that they refer to as PABs - but there is no indication that PABs form in other cell types or tissues, or in other contexts, so it is unclear whether PABs are an important structure or an artifact of the system. Furthermore, part of the motivation of the work seems to be to understand MFN-related pathologies, but using a MEF system does not necessarily allow for that. One way to strengthen this part of the manuscript is to potentially use disease-relevant MFN2 mutations and determine downstream effects on cell morphology and migration.

    State what audience might be interested in and influenced by the reported findings.

    • This work would appeal to card-carrying cell biologists.

    Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    • cell migration; actin; mitochondria
  6. Version published to 10.1101/2022.10.03.510661 on bioRxiv