Dear Dr. Lautenschläger,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Florian Rehfeldt

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1:  The authors have submitted a decently written manuscript describing the effects of a new compound miuraenamide A (MiuA), recently found to affect actin dynamics, on focal adhesions, cell contractility and migration. They show that in cell culture the treatment with MiuA in two cell types reduce average focal adhesion size but increase the number of adhesions. They also find that the strength of these adhesions, despite being higher in number, are less adhesive. The authors also show that amoeboid type migration is unaffected by MiuA treatment. Overall the experiments seem to be well-performed and nearly adequate analysis. However, the overall writing style needs some adjustment for clarity as well as some additional questions need to be addressed before this paper should be accepted for publication in PONE. The following issues should be addressed:

(1) The way the manuscript is written seems very redundant in many places. For example, the Introduction section is extremely long and is written to include the results. Provide only a quick summary of your results (not going figure by figure) in the introduction and keep the results section that you do have for the detailed explanation of the figures. On the first read of the manuscript, I thought that the introduction’s run through of the figures was indeed the results section.

(2) The authors use two cell types for the beginning two figure’s worth of experiments but then drop the use of the MEF cells. Why is this? MEF cells are commonly used for cell migration experiments, especially in 2D settings, so some analysis of their behavior upon MiuA treatment would be helpful to the field and further the impact of this paper.

(3) On a similar note, the authors don’t seem to address 2D migration parameters, which would be greatly beneficial to the broader cell migration community (and again, boost the impact of this publication). I suggest that the authors analyze the effect of MiuA treatment on 2D migration parameters such as speed, persistence, directionality, etc. These parameters are only analyzed in the context of the PDMS confinement channels during amoeboid migration. Is it known how MiuA affects unconfined 2D migration? Such a change in focal adhesions parameters surely affects 2D migration.

(4) In Figure 1, the authors show only the spreading area and actin filament length analysis for MEF cells but not RPE-1 cells – why? There seems to be a significant change in the spreading area for RPE-1 cells based on the images in panel (1C). Since the authors rely heavily on RPE-1 cells throughout the manuscript, this should not be omitted here.

Reviewer #2:  The authors demonstrate the effect of an actin-stabilizing agent (MiuA) on the adhesivity, contractility and migration of two cell types. They show that although focal adhesions are impacted by this drug, the contractility and ability to migrate in the amoeboid mode are not.

These findings are an interesting addition to a growing body of work on cell mechanics and cell-substrate mechanical interactions. However, some more mechanistic insights and explanation of the data could strengthen this study, and some of the rationale is not well-described. Some specific concerns and questions are:

1. The Introduction paragraphs 3 and 4 are essentially a summary of the Results, which is unconventional. As a reader, I am interested in knowing more about the rationale and background of the study. what is known about the biochemical mechanism of how MiuA works: does it inhibit actin polymerization or severing, for example? how does it interact with actin binding proteins? what are the pros and cons of using this as a stabilizng agent vs. jusplakinolide or phalloidin? what are the RPE1 and MEF cell types and why are they the suitable candidates in this study?

2. In Fig. 1, it is abrupt to start with quantification before showing cell images first. Also, why quantification is shown only for MEF cell type only in Figs 1a and 1b and not for RPE1?

3. Fig. 1c focuses on individual cells, but to be convincing the authors should also show a wider field of view with many cells, so that the reader can see the representative behavior.

4. The MiuA treated MEF cell shown has an interesting spindle-like morphology. Have the authros tried to quantify this effect? A quantification of the cell aspect ratio would reflect this, for example. It will be interesting to see how cell morphology is affected in addition to cell area, by MiuA.

5. There should be some explanation, at least qualitative, why the contractility is not affected although adhesivity is.

6. Why are neutrophils intriduced for motility measurements, and not MEF? do these not migrate?? there could be a cartoon to illustrate how persistence is measured. In addition to adhesions, can the 1D vs 2D nature of the migration also play a role?

7. Similar to Introduction, the Conclusion could be stronger if there was some discussion of the broader context of this work. How does jusplakinolide affect these quantities: cell adhesivity, contractility, and migration for example?

8. The images provided are low resolution at least in the reviewer's copy. Perhaps the images analyzed should also be shared in high resolution alongside the supplementary data.

9. There is some informal/colloquial use of English in some places that seem out of place in a technical document. Sentences should not begin with "And" as in line 63 or 88, and "very well" is not a quantifiable statement in line 76, just to take two examples. The authors could use better human proofreading in addition to using AI-based tools which are not often adequate for this purpose.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Dear Dr. Lautenschläger,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript was substantially improved by the revision and addressing the issues of both referees. However, before acceptance for publication it is imperative to address the last open points raised by referee 2.

Please submit your revised manuscript by Aug 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Florian Rehfeldt

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors have satisfied all of my concerns. I recommend an acceptance of this manuscript for publication.

Reviewer #2: The authors have significantly improved the clarity of the manuscript in this revised version. They have now answered most of my questions, but I have some more remaining comments which I believe should be addressed before final acceptance.

1.The authors explain that the some of the quantification for RPE-1 cells, corresponding to the results in Fig. 1, were carried out in their previous publication, Ref.22. However, in that study, the MiuA-treated RPE-1 cells increased their spread area, whereas MiuA-treated MEF cells showed no change in spread area. They think this puzzling result is because different doses of MiuA were used for the two cell lines. This raises the obvious question: why not do the same dosage for both cell lines to establish this? Is there any practical limitation to this?

2. Related to above, the authors discuss that one of the disadvantages of using jasplakinolide is that its effect is highly dosage dependent. However, they also found that MiuA's effect is also dosage dependent. How sensitive is this dosage dependence? Have they tried for example, different doses of MiuA on RPE-1 or MEF?

3. In Fig. 1a, the MiuA treated MEF cells shown look distinctly smaller by eye than the control MEF cells. Why is this? Is this image not a representative sample of the whole population of cells? In this case, it might be more convincing to choose a different image, or show additional images in the SI.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

Modulation of Cellular Adhesion, Contractility, and Migration by MiuA: A Comprehensive Analysis of Its Biomechanical Impact

PONE-D-24-57233R2

Dear Dr. Lautenschläger,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Florian Rehfeldt

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: The authors have shown additional data and satisfactorily answered my remaining questions about dosage dependence of miuA and additional cell images. I think this is technically sound and interesting work, and is ready to be accepted for publication.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: No

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