Laminin N-terminus α31 regulates corneal epithelial cell adhesion and migration through modifying the organization and proteolytic processing of laminin 332

Lee D. Troughton; Valentina Iorio; Liam Shaw; Conor J. Sugden; Natasha D. Chavda; Peter Wilson; Djamilla Simoens; Andrea E. Conway; Stefano Sala; Simon Kaja; Kazuhiro Yamamoto; Kevin J. Hamill

doi:10.1371/journal.pone.0338797

Peer Review History

Original SubmissionAugust 20, 2025
20 Aug 2025 Author Response Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. https://doi.org/10.1371/journal.pone.0338797.r001
22 Oct 2025 Decision Letter - Alexander Ljubimov, Editor Dear Dr. Hamill, 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. Please submit your revised manuscript by Dec 06 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, Alexander V Ljubimov, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. Please include a complete copy of PLOS’ questionnaire on inclusivity in global research in your revised manuscript. Our policy for research in this area aims to improve transparency in the reporting of research performed outside of researchers’ own country or community. The policy applies to researchers who have travelled to a different country to conduct research, research with Indigenous populations or their lands, and research on cultural artefacts. The questionnaire can also be requested at the journal’s discretion for any other submissions, even if these conditions are not met. Please find more information on the policy and a link to download a blank copy of the questionnaire here: https://journals.plos.org/plosone/s/best-practices-in-research-reporting. Please upload a completed version of your questionnaire as Supporting Information when you resubmit your manuscript. 3. Thank you for stating in your Funding Statement: “This work was supported by funding from the Biotechnology and Biological Sciences Research Council (K.H BB/L020513/1 and BB/P025773/1, and a BBSRC DTP PhD studentship), Northwest Cancer Research Fund (K.H), Fight For Sight (K.H New lecturers' award), British Skin Foundation (K.H PhD studentship), Foundation for the Prevention of Blindness (K.H), ECMage impact award (K.H), Versus Arthritis Career Development Fellow (K.Y 21447), Versus Arthritis Bridging Fellow (K.Y 23137). Additional support from the National Institute of Health (S.K grant R24/EY032440), the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (S.K), the Richard A. Perritt M.D. Charitable Foundation and the Illinois Society for the Prevention of Blindness are gratefully acknowledged (L.T). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. https://www.ukri.org/councils/bbsrc/ https://www.fightforsight.org.uk/ https://www.britishskinfoundation.org.uk/ https://nwcr.org/ https://versusarthritis.org/ https://www.nih.gov/ https://eyehealthillinois.org/” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 4. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.] Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: “This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BB/L020513/1 and BB/P025773/1, and a BBSRC DTP PhD studentship), Northwest Cancer Research Fund, Fight For Sight (New lecturers' award), British Skin Foundation (PhD studentship), Foundation for the Prevention of Blindness, ECMage impact award, Versus Arthritis Career Development Fellow (21447), Versus Arthritis Bridging Fellow (23137). Additional support from the National Institute of Health (grant R24/EY032440), the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology, the Richard A. Perritt M.D. Charitable Foundation and the Illinois Society for the Prevention of Blindness are gratefully acknowledged.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “This work was supported by funding from the Biotechnology and Biological Sciences Research Council (K.H BB/L020513/1 and BB/P025773/1, and a BBSRC DTP PhD studentship), Northwest Cancer Research Fund (K.H), Fight For Sight (K.H New lecturers' award), British Skin Foundation (K.H PhD studentship), Foundation for the Prevention of Blindness (K.H), ECMage impact award (K.H), Versus Arthritis Career Development Fellow (K.Y 21447), Versus Arthritis Bridging Fellow (K.Y 23137). Additional support from the National Institute of Health (S.K grant R24/EY032440), the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (S.K), the Richard A. Perritt M.D. Charitable Foundation and the Illinois Society for the Prevention of Blindness are gratefully acknowledged (L.T). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. https://www.ukri.org/councils/bbsrc/ https://www.fightforsight.org.uk/ https://www.britishskinfoundation.org.uk/ https://nwcr.org/ https://versusarthritis.org/ https://www.nih.gov/ https://eyehealthillinois.org/” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 8. 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. Additional Editor Comments (if provided): The paper has been reviewed and some minor concerns voiced. Please revise the paper according to the critique. 1. Please revisit Figure 4F-G (lines 234-236) and add missing Fig.4G. 2. “For gelatin zymography assays, concentrated normalized conditioned media…” Please specify how it was normalized. 3. It is customary to call skin epithelial cells keratinocytes. However, corneal counterparts are called corneal epithelial cells. 4. Adenovirus has been shown to be toxic for cultured primary human limbal epithelial cells. Please confirm that no toxicity at the used doses was observed (like with Apopnexin assay). 5. Whereas MMP-9 was mostly present in the pro-form, MMP-2 showed clear activation after LaNt�31 addition. Please discuss. 6. Please explain in more detail how the zymograms were obtained. Typically, the gel is stained with Coomassie, then incubated with the extracts/media and the enzyme bands are seen as white against the blue background. Here, the bands are black against white background. Was the picture color-reversed or another method used? 7. Supplemental figure 1. It is unusual to see K12 expression on hTERT-immortalized HCEC. Please comment that they could be more differentiated as compared to primary limbal cells. 8. For readers not familiar with HD structure, please mention that the used proteins, Type XVII collagen and BPAG, are HD structural components and markers. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The data presented characterize LaNt α31 as a regulator of epithelial homeostasis via modulation of cell–matrix adhesion and LM332 architecture and suggest that LaNt α31 could serve as a molecular switch from migratory to adhesive epithelial conditions. The results obtained were presented in clear and well-documented manner, with the exception of the paragraph describing increased co-localisation of β4 integrin with type XVII collagen and BPAG1e rescued by MMP inhibition but not by non-MMP inhibitors (Figure 4F-G) (lines 234-236), as there was no Fig.4G presented in the manuscript. ****** 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 ******** [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0338797.r002
Revision 1
21 Nov 2025 Author Response We appreciate all the reviewer’s comments and have addressed each point for your consideration, below. 1. Please revisit Figure 4F-G (lines 234-236) and add missing Fig.4G. We have corrected this issue, adding panel 4G to figure 4. 2. “For gelatin zymography assays, concentrated normalized conditioned media…” Please specify how it was normalized. After collecting condition media, the cells were fixed and DAPI-stained nuclei counted. Media volume was then adjusted based on that count. We have described this in the method section and have also added it to the figure legend for figure 4. 3. It is customary to call skin epithelial cells keratinocytes. However, corneal counterparts are called corneal epithelial cells. We agree and have changed keratinoyctes to corneal epithelial cells throughout. 4. Adenovirus has been shown to be toxic for cultured primary human limbal epithelial cells. Please confirm that no toxicity at the used doses was observed (like with Apopnexin assay). We appreciate this suggestion. hTCEpi are a limbal-derived cell line, immortalised, more mature epithelial cells. We have not experienced any evidence of toxicity using adenovirus with this line. Throughout, we have used a GFP-only adenovirus as a viral transduction and protein expression control. The GFP-virus conditions displayed no significant difference to the non-transduced hTCEpi in any experiments. 5. Whereas MMP-9 was mostly present in the pro-form, MMP-2 showed clear activation after LaNta31 addition. Please discuss. We have included supplemental figure 5, which provides all our raw blots and gelatin zymography images. You can perhaps better appreciate here that we see an increase in activity for MMP2 and MMP9. The currently literature is not definitive on whether laminin LG3/4 linker is cleaved by MMP2 or MMP9, although both appear to be capable, therefore we are deliberately not making a statement along those lines. We have, however, stated in the discussion that future should focus on investigating the relationship between LaNt α31’s interaction with LM332 and MMPs. 6. Please explain in more detail how the zymograms were obtained. Typically, the gel is stained with Coomassie, then incubated with the extracts/media and the enzyme bands are seen as white against the blue background. Here, the bands are black against white background. Was the picture color-reversed or another method used? You are correct, this is an inverted image as we believe the contrast allows for better visualisation. We have now clarified this in the figure legend and indicated in figure 4 panel A (and supplemental figure 5) itself to highlight the inversion. The zymography methods are described in the method section. 7. Supplemental figure 1. It is unusual to see K12 expression on hTERT-immortalized HCEC. Please comment that they could be more differentiated as compared to primary limbal cells. To our knowledge, these cells express both keratin 3/12 and 5/14, although it is true that they likely have higher expression of 5/14 as they are not stratified. We deliberately hadn’t commented on the differentiation state of these cells as we haven’t looked in detail at their keratin expression profile. We included this figure as supplement to demonstrate they are keratin positive epithelial cells. We have however added a line in 112/113 stating they appear to be a more mature corneal epithelial line. 8. For readers not familiar with HD structure, please mention that the used proteins, Type XVII collagen and BPAG, are HD structural components and markers. Thank you for pointing this out. With have included this in the results section, line 188. Additionally, we have removed the funding body information from the acknowledgements and amended the funding disclosure statement as requested, uploading separately as “Funding statement”. We have also compiled all the numerical data used in a single excel file which will be made available. All the uncropped blots and gel images have been included in the manuscript in a new figure (Supplemental Figure 5). Thank you for your time and consideration. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0338797.r003
27 Nov 2025 Decision Letter - Alexander Ljubimov, Editor Laminin N-terminus α31 regulates corneal epithelial cell adhesion and migration through modifying the organization and proteolytic processing of laminin 332. PONE-D-25-45306R1 Dear Dr. Hamill, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Alexander V Ljubimov, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0338797.r004
Formally Accepted
Acceptance Letter - Alexander Ljubimov, Editor PONE-D-25-45306R1 PLOS One Dear Dr. Hamill, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Alexander V Ljubimov Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0338797.r005

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .