Alternative method to visualize receptor dynamics in cell membranes

Ravelli Cosetta; Corsini Michela; Ventura Anna; Domenichini Mattia; Grillo Elisabetta; Mitola Stefania

doi:10.1371/journal.pone.0304172

Peer Review History

Original SubmissionFebruary 5, 2024
3 Apr 2024 Decision Letter - Eleftherios Paschalis Ilios, Editor PONE-D-24-04439Alternative method to visualize receptor dynamics in cell membranes.PLOS ONE Dear Dr. Mitola, 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 May 18 2024 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. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: The two reviewers made valid comments which need to be addressed before this paper is considered further. Please review the comments and address them to the best of your ability. Thank you [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Partly Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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: N/A Reviewer #2: Yes ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: 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: The proposed manuscript by R. Cosetta and al. is dedicated to the development of a new approach to determine the receptor dynamics in the cell membrane. This approach was developed to use technology easily available in most research centers. This technological manuscript well describes the interest of such approach. However, some important points should be addressed to improve this manuscript: - The authors indicated that this approach was "...an economical and simple assay to follow and characterize membrane receptor dynamics..." and that the use of epifluorescence microscopy (EM) was possible. However, in their descriptions, the authors used confocal microscopy and some related techniques such as image stacking (not really accessible in EM). The authors should describe the use of EM in this approach. - The number of cells analyzed was not reported. The number of experiments was also not reported. These items should be reported. - How was the percentage of positive areas determined? Image analysis ? Software used ? procedure ? - The authors do not discuss other methods to study receptor dynamics. This should be discussed. The advantages and limitations of this new approach should also be discussed. - The authors cited reference 17 for analysis of VEGFR and other receptor expression, but this reference did not use the same method. This should be discussed. - The authors do not indicate whether the internalization of the receptors could be detected with this method ? or not. Reviewer #2: The authors provide an alternative cost effective method for visualizing receptor dynamics in cell membranes.The protocol is well-described, providing clear step-by-step instructions for conducting the experiments. There are few suggestions that would help to improve the manuscript: 1. The introduction needs to be more general rather than focusing on one type of membrane receptors. 2. Figure 1: Experimental layout - the steps shall be described in details with graphical/pictorial representation of steps making it easier for the readers to follow. 3. Authors should incorporate the practical problems faced and troubleshooting they performed. 4. Lastly, it would be good to add a table showing the limitations and advantages of the existing techniques including the one described by the authors. ****** 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: Yes: Alain COUVINEAU 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. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0304172.r001
Revision 1
24 Apr 2024 Author Response We have addressed reviewers’ criticisms (in italics) as it follows: 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes We thank reviewers for appreciating the utility of our protocol and for recognizing its novelty. 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io (DOI: dx.doi.org/10.17504/protocols.io.rm7vzj8z8lx1/v1 (Private link for reviewers: https://www.protocols.io/private/46322724FDB111EEB7ED0A58A9FEAC02 to be removed before publication.) in the Materials and Methods section of the manuscript or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Partly Reviewer #2: Yes Following the Editor suggestion the step-by-step protocol, included in Supporting information in the previous version, was included and published into protocols.io . Please see: Please see DOI: dx.doi.org/10.17504/protocols.io.rm7vzj8z8lx1/v1 (Private link for reviewers: https://www.protocols.io/private/46322724FDB111EEB7ED0A58A9FEAC02 to be removed before publication.) 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes Here we described immobilized VEGF-A-recruited receptor to demonstrate the cogency of our method. The method allowed us to compare different experimental conditions such as different stimuli, inhibitors, receptors mutants and multiple receptors and co-receptors at the same time (see Damioli et al.). 4. If the manuscript contains new data, have the authors made this data 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: N/A Reviewer #2: Yes We added statistic detail s in figure captions. 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes The typos and grammar errors were corrected. Reviewer Comments to the Author: Reviewer #1 The proposed manuscript by R. Cosetta and al. is dedicated to the development of a new approach to determine the receptor dynamics in the cell membrane. This approach was developed to use technology easily available in most research centers. This technological manuscript well describes the interest of such approach. However, some important points should be addressed to improve this manuscript: - The authors indicated that this approach was "...an economical and simple assay to follow and characterize membrane receptor dynamics..." and that the use of epifluorescence microscopy (EM) was possible. However, in their descriptions, the authors used confocal microscopy and some related techniques such as image stacking (not really accessible in EM). The authors should describe the use of EM in this approach. Working in Z-stack and quantifying fluorescence in each slice, it is important to have a good 3D sectioning. We achieved it using structured illumination technology which is less expensive and time-consuming than a confocal laser microscopy. Alternatively a deconvolution algorithm ( plugins for Fiji or DeconvolutionLab and DeconvolutionLab2 (EPFL) (https://bigwww.epfl.ch/deconvolution/) can be used to analyze fluorescent stack image series . See lines 196-202 and Table 1 - The number of cells analyzed was not reported. The number of experiments was also not reported. These items should be reported. We apologize for forgetting to include the number of analyzed cells in each experiment and the number of experiments carried out. For each experiment, repeated three times, 20 cells were analyzed at each time point. We added this information in the Expected Results section (see linse 161-162). - How was the percentage of positive areas determined? Image analysis? Software used? procedure? All the information regarding image quantification is reported in Supporting information: Step 5, Image quantification and data analysis (day 5). Briefly, for each experiment images were acquired with the same exposure time. Then, Z-stacks were analyzed with Fiji software. After defining a threshold, we drew a ROI for each cell in which we quantified the number of fluorescence-positive pixels in every slice of the Z-stack. Finally, we calculated the percentage of the receptor present in every slice to visualize its distribution. - The authors do not discuss other methods to study receptor dynamics. This should be discussed. The advantages and limitations of this new approach should also be discussed. We thank both Reviewers for the suggestion of deepening advantages and limitations of the methods currently available to study receptor dynamics on cell membranes. To address it, we decided to include a Table in Discussion section (Table 1, line 222) describing positive and negative aspects of the proposed protocol compared with the other two main methodologies: FRAP and SPT. - The authors cited reference 17 for analysis of VEGFR and other receptor expression, but this reference did not use the same method. This should be discussed. In the manuscript we refer to the papers by Damioli et al. (DOI) and Ravelli et al. (DOI) in which this protocol was used. In those publications, however, we focused our attention only on the quantification of receptors (VEGFR2 and Beta3 integrin) recruited in close contact with the immobilized factors. Here we analyzed the distribution, in time, of the entire pool of receptor. - The authors do not indicate whether the internalization of the receptors could be detected with this method ? or not. We added a comment on the possibility of detection receptor internalization in lines 218-222. Reviewer #2: The authors provide an alternative cost effective method for visualizing receptor dynamics in cell membranes. The protocol is well-described, providing clear step-by-step instructions for conducting the experiments. There are few suggestions that would help to improve the manuscript: 1. The introduction needs to be more general rather than focusing on one type of membrane receptors. We introduced the different classes of membrane receptors in the introduction section ( see lines 32-36) , Then we focused only on one pass transmembrane receptors. Although we have no reason to think that the described protocol cannot be used to follow all kinds of receptors, we have alway applied it to analyze the dynamics of single-pass receptors ( e.i tyrosine kinase and integrins). 2. Figure 1: Experimental layout - the steps shall be described in details with graphical/pictorial representation of steps making it easier for the readers to follow. Following the reviewer’s suggestion we included in the revised version (Fig 2) a cartoon which recapitulates the steps from the coverslip flipping to image quantification. Fig 2: Cartoon of steps 3-5 of experimental flow chart (see lines 89, 147 and Fig2). 3. Authors should incorporate the practical problems faced and troubleshooting they performed. The practical problem and the critical steps were included into DOI: dx.doi.org/10.17504/protocols.io.rm7vzj8z8lx1/v1 (Private link for reviewers: https://www.protocols.io/private/46322724FDB111EEB7ED0A58A9FEAC02 to be removed before publication.) 4. Lastly, it would be good to add a table showing the limitations and advantages of the existing techniques including the one described by the authors. We included limitations and advantages in Table 1 (Advantages and disadvantages of microscopy techniques for the study of molecule relocation) and described in the Discussion section of the revised version. See Table 1, line 222 . Sincerely Prof.ssa Stefania Mitola Attachments Attachment Submitted filename: rebuttal.pdf https://doi.org/10.1371/journal.pone.0304172.r002
8 May 2024 Decision Letter - Eleftherios Paschalis Ilios, Editor Alternative method to visualize receptor dynamics in cell membranes. PONE-D-24-04439R1 Dear Dr. Mitola, 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. 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Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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 Reviewer #2: Yes ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: 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: The proposed manuscript by R. Cosetta and al. is dedicated to the development of a new approach to determine the receptor dynamics in the cell membrane. This approach was developed to use technology easily available in most research centers. This technological manuscript well describes the interest of such approach. The authors having totally replied to my previous comments. In this context, the present manuscript should be accepter for publication. Reviewer #2: The authors have successfullly addressed all the comments and revised the manuscript accordingly. ****** 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: Yes: Alain Couvineau Reviewer #2: Yes: Jyoti Sharma ******** https://doi.org/10.1371/journal.pone.0304172.r003
Formally Accepted
31 May 2024 Acceptance Letter - Eleftherios Paschalis Ilios, Editor PONE-D-24-04439R1 PLOS ONE Dear Dr. Stefania, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Eleftherios Paschalis Ilios Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0304172.r004

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