PONE-D-23-13577Interfering with the ERC1–LL5β interaction disrupts plasma membrane–associated platforms and affects tumor cell motilityPLOS ONE

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"We thank the personnel of the Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC) of the San Raffaele Scientific Institute for technical support, Prof. Isabella Felli (University of Florence, Italy) for helpful discussion,  and Dr. Yuko Mimori-Kiyosue (RIKEN Center for Biosystems Dynamics Research, Kobe, Japan) for the LL5� antibody. IdC was supported by a grant from AIRC—Associazione Italiana per la Ricerca sul Cancro (IG 2017 Id.20203). KS and MR were supported by postdoctoral fellowships from AIRC. LMR was supported by a FCSR-Fronzaroli fellowship; work performed by LMR was in partial fulfilment of the requirements for obtaining the PhD degree at the Vita-Salute San Raffaele University, Milano, Italy. This work benefited from access to the Protein Production in E. coli with Isotope Labelling for NMR platform at CERM/CIRMMP (Florence, Italy), an Instruct-ERIC centre. Financial support was provided by Instruct-ERIC (PID: 17416). "

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Additional Editor Comments:

Dear Dr. Ivan de Curtis,

Thank you for submitting your manuscript to PLOS One. Your work has now been evaluated by two referees and both of them showed great interest in the story. There are, however, a few concerns that need to be addressed before we can move forward. I hope that you find these comments helpful to improve the manuscript.

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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?

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

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 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

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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

Reviewer #2: Yes

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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 submitted manuscript written by Ivan de Curtis et al. analyses extensively the interacting sites of the ERC1–LL5β binding. The manuscript is overall well and clearly written, I really liked the proposed idea that LL5β fragment inhibiting the interaction between ERC1 and LL5β leads to the prevention of the complex formation of these adaptor proteins and interferes with the processes required for tumour cell invasion.

I have some suggestions for improving the manuscript, as in this form has some insufficiency:

1., Several interesting topics come up during the results part which are not mentioned earlier and introduced properly. Please insert some sentences in the introduction about these topics:

-Differences and similarities of LL5β and LL5alfa regarding their effects, functions, and structure.

-How LL5β serve as a bridge between the actin cytoskeletal system and the microtubule system in cells and regulates via partner molecules both filament nets?

-Molecular structure (domains) of ERC1.

-Insert a sentence describing the differences between lamellipodia and invadopodia.

2., I would include an overview about the already published results (or search in databases) about the expression level changes of ERC1 and LL5β in cancers (especially it would be most relevant in breast cancer, as the MDA-MB-231 is a human breast adenocarcinoma cell line used in this study). This would fit at the very end of the results part.

3., Need reference for this sentence: Line 364: “In COS7 cells the endogenous LL5 protein is represented mostly by LL5alfa”

4., I would suggest to perform a PLA assay (Duolink) in a cell line expressing endogenous LL5β and endogenous ERC1. In this setting, it would be very convincing to show that the introduction of LL5β(381-510) disrupts the PLA signal (for endogenous full-LL5β and endogenous ERC1). Because the manuscript states that: “line 580: These findings indicate that the ERC1-binding fragment LL5β(381-510) interferes specifically with the interaction between the ERC1 and LL5 full length proteins.” Actually, the transduced LL5β full length protein was measured not the endogenous full length LL5β proteins in the cancer cells.

5., Anyway, it is not clear if the MDA-MB-231 cell express or not the endogenous LL5β full length protein?

Reviewer #2: This manuscript deals with ERC1-LL5beta interaction in breast cancer cells. In particular, the binding sites of these molecules and the inhibitory effects of LL5beta fragments were researched well. However, some data and description are missing

Major points:

1. In the breast cancer motility, the author showed ERC1 accumulation in PMAP. And,

the fragment of ERC1 inhibits the accumulation of ERC1. The author mentioned that the inhibition of accumulating ERC1 in PMAP decreases the cell motility. However, the molecular function of ERC1 in cell migration is not mentioned. The author should mention the mechanism and show the morphological change of the cell and decreasing the any other PMAP components by adding the fragments.

2. The formation of invadopodia is multistep process. The function of ERC1 in forming the invadopodia is not mentioned. Which molecules do ERC1 participate in the formation of invasive protrusions?　How signal is the pathway induced by ERC1? The author needs to describe. In addition, the author should perform the experiments showing the invadopodial signal transduction including ERC1 and inactivating by adding fragments.

3. In quantitative evaluation of invadopoaia, the author used invadopodial number par

area. As far as I know, usually, the gelatin degeneration area per cell is correlate the invasive ability. Why the author used the unit “ invadopodial number per” ?

4. If full length-LL5beta can make dimer/oligomer and the fragment can not, the GFP (with fulllength/fragment LL5beta) density of the ERC1 accumulating legion may be different. Therefore, the correlation of GFP and ERC1 intensity should be measured and compared.

Minor points:

1. In the figure of immunoblotting, the author described the weight of samples.

Is it exact, and important?

2. In the experiments of cell migration, the typical trajectories of control and should be shown.

3. The reference to GFP (305-450) and GFP(381-510) in Figure 6 is misleading and should be corrected to LL5β-GFP ().

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Reviewer #1: No

Reviewer #2: No

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Interfering with the ERC1–LL5b interaction disrupts plasma membrane–associated platforms and affects tumor cell motility

PONE-D-23-13577R1

Dear Dr. de Curtis,

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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Kind regards,

Zhiming Li, Ph.D.

Academic Editor

PLOS ONE

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

Reviewer #2: (No Response)

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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

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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 points raised during the review process were addressed and all the corrections were made. This manuscript is suitable for accepting in PLOS ONE and the publishing process can move forward.

Reviewer #2: I apologize for the delay in the review of this revised manuscript. The author was already addressed my question and proposal.

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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

Reviewer #2: No

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