Assembly assay identifies a critical region of human fibrillin-1 required for 10–12 nm diameter microfibril biogenesis

Sacha A. Jensen; Ondine Atwa; Penny A. Handford

doi:10.1371/journal.pone.0248532

Peer Review History

Original SubmissionNovember 2, 2020
24 Nov 2020 Decision Letter - Maria Gasset, Editor PONE-D-20-34409 Assembly assay identifies a critical region of human fibrillin-1 required for 10 - 12 nm diameter microfibril biogenesis PLOS ONE Dear Dr. Sacha Jensen, Thank you for submitting your manuscript to PLOS ONE. First of all my personal apologies in the delay in getting back to you. Several reviews declined the invitation. Given that the single review is critic but positive and constructive, we invite you to submit a revised version of the manuscript that addresses all the indicatec points Please submit your revised manuscript by january 15 2021. 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. Please include the following items when submitting your revised manuscript: 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. 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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. [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? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript by Jensen et al. describes important functional differences how mutations in fibrillin-1 lead to the classical Marfan syndrome and the more severe neonatal Marfan syndrome. The authors tested a series of mutations in both groups using a mini-fibrillin-1 construct transfected in the fibroblastic cell line MSU-1.1 to analyze secretion. The same mutations were introduced in full-length GFP-tagged constructs and expressed in 293 cells. These cells secrete recombinant fibrillin-1, but do not assemble them. Therefore, the authors use a previously established co-culture system with human dermal fibroblasts to test extracellular assembly of the mutant fibrillin-1 constructs. Most cMFS and nMFS mutations were secreted into the culture medium. While all cMFS mutations assembled into typical microfibrils, the nMFS mutations consistently did not assemble. This is novel and exciting, because it defines a new region (the neonatal region) as a critical determinant in microfibril assembly. The experiments are convincing and well controlled. I have the following comments. Major comments In the model (Fig. 5), it is a bit confusing what the authors mean with lateral assembly. I think they mean the lateral interactions between the ~8 molecules within one single microfibril. But please keep in mind that there are also lateral interactions described in the literature between individual microfibrils to produce bundles of microfibrils. The confusion stems from Fig. 5Biii where a second microfibril is indicated in the last step that is blocked by nMFS mutations. It gives the impression that the lateral bundling between microfibrils is affected. But I think this is not what the authors mean. Also in A, step 4, it does not become entirely clear from the schematic how the green neonatal region becomes positioned at the bead region. A better explanation and color coded individual fibrillin-1 molecules would help to better understand the proposed model. It was shown in the literature (PMID 21784848) that neonatal mutations affect the ability of mutant fibrillin-1 constructs to interact with heparin (and presumably with heparan sulfate). However, this is not well incorporated into the model. In A, the heparan sulfate chains between #3 and #4 do not interact with these moieties. Is there any support from the literature or from the authors’ experiments that the neonatal region can physically interact with the bead region? Since there are several fibrillin-1 assembly models available, it would be useful to discuss how the data fit (or not) with other assembly models, ideally trying to reconcile the various models. So overall, the model requires significantly more work and refining to incorporate the existing literature and to provide a clearer representation. Minor comments The authors use Fbn1 as an abbreviation for human fibrillin-1 protein. As per accepted gene and protein nomenclature, human protein should be abbreviated FBN1. ****** 6. PLOS authors have the option to publish the peer review history of their article (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.] 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. 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Revision 1
18 Jan 2021 Author Response Major comments In the model (Fig. 5), it is a bit confusing what the authors mean with lateral assembly. I think they mean the lateral interactions between the ~8 molecules within one single microfibril. But please keep in mind that there are also lateral interactions described in the literature between individual microfibrils to produce bundles of microfibrils. The confusion stems from Fig. 5Biii where a second microfibril is indicated in the last step that is blocked by nMFS mutations. It gives the impression that the lateral bundling between microfibrils is affected. But I think this is not what the authors mean. Also in A, step 4, it does not become entirely clear from the schematic how the green neonatal region becomes positioned at the bead region. A better explanation and color coded individual fibrillin-1 molecules would help to better understand the proposed model. - Figure 5 has now been updated to take into account the reviewer's comments. In panel B, where we depict the lateral assembly that we propose is driven by the neonatal region, we have provided more details on the final assembly step (4) shown in panel A. We also now show a second set of fibrillin monomers in grey, aligned head-to-tail, and how these microfibril precursors may interact laterally to produce the mature structure. It was shown in the literature (PMID 21784848) that neonatal mutations affect the ability of mutant fibrillin-1 constructs to interact with heparin (and presumably with heparan sulfate). However, this is not well incorporated into the model. In A, the heparan sulfate chains between #3 and #4 do not interact with these moieties. - Panel A of figure 5 has been simplified to reflect the interactions between heparan sulphate and specific sites on fibrillin-1. Only 3 of the 7 known heparan interaction sites, at the N- and C- termini and the neonatal region, have been shown for clarity. Is there any support from the literature or from the authors’ experiments that the neonatal region can physically interact with the bead region? - The neonatal region of fibrillin-1 is not currently known to interact with any other fibrillin-1 domains. It may be that earlier assembly steps are needed before the required spatial arrangement of domains is created in the nascent microfibril to allow an interaction with the neonatal region. We have therefore added a comment in the discussion (p.20, line 4) to address this. Since there are several fibrillin-1 assembly models available, it would be useful to discuss how the data fit (or not) with other assembly models, ideally trying to reconcile the various models. - We have now included a paragraph that addresses this point at the end of p.19 of the discussion, describing how our model for the role of the neonatal region in assembly is more compatible with a staggered model of microfibril organisation. Minor comments The authors use Fbn1 as an abbreviation for human fibrillin-1 protein. As per accepted gene and protein nomenclature, human protein should be abbreviated FBN1. - This has now been corrected. https://doi.org/10.1371/journal.pone.0248532.r002
19 Feb 2021 Decision Letter - Maria Gasset, Editor PONE-D-20-34409R1 Assembly assay identifies a critical region of human fibrillin-1 required for 10 - 12 nm diameter microfibril biogenesis PLOS ONE Dear Dr. Sacha Jensen, Thank you for submitting your revised manuscript to PLOS ONE and include all indicated amendements. However before its acceptance a minor correction must be performed. Define fibrillin-1 abbreviation as FBN1 after the first time fibrillin-1 is mentioned and substitute the full name along the rest of the text and in figures (plots and legends). Please submit your revised manuscript by february 20th. 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. Please include the following items when submitting your revised manuscript: 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Maria Gasset, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: All critiques have been appropriately addressed. ****** 7. PLOS authors have the option to publish the peer review history of their article (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.] 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. https://doi.org/10.1371/journal.pone.0248532.r003
Revision 2
25 Feb 2021 Author Response Dear Editor, Thank you for the recent response to our manuscript, “Assembly assay identifies a critical region of human fibrillin-1 required for 10 - 12 nm diameter microfibril biogenesis” (manuscript number: PONE-D-20-34409R1). We were asked to make a minor correction to the manuscript before its acceptance, which was to define the fibrillin-1 abbreviation as FBN1 after the first time fibrillin-1 is mentioned and to then substitute the full name through the rest of the article (including figures and legends). This has now been done, with the FBN1 abbreviation being defined at its first mention in the abstract. A few other minor typographical corrections have also been made, including the renaming of the recombinant GFP-tagged version of fibrillin-1 from "GFP-FBN" to the more specific "GFP-FBN1". In addition, after checking the figures using the PACE digital diagnostic tool, it was noted that figure 4 did not meet PLOS requirements and has therefore been divided into two new figures. All changes are highlighted in the marked-up version of the manuscript. We would also like to make a change to our financial disclosure statement to acknowledge funding from the UK Medical Research Council (MRC), which contributed to the work in this manuscript. The updated statement would read: SAJ and PAH gratefully acknowledge support from Arthritis Research UK (www.versusarthritis.org; project grant no. 20785) and the MRC (https://mrc.ukri.org; grant number MR/M009831/1). Yours sincerely, Sacha Jensen 25/2/21 https://doi.org/10.1371/journal.pone.0248532.r004
1 Mar 2021 Decision Letter - Maria Gasset, Editor Assembly assay identifies a critical region of human fibrillin-1 required for 10 - 12 nm diameter microfibril biogenesis PONE-D-20-34409R2 Dear Dr. Sacha Jensen, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Maria Gasset, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0248532.r005
Formally Accepted
10 Mar 2021 Acceptance Letter - Maria Gasset, Editor PONE-D-20-34409R2 Assembly assay identifies a critical region of human fibrillin-1 required for 10 - 12 nm diameter microfibril biogenesis Dear Dr. Jensen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Maria Gasset Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248532.r006

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