TAAR8 Mediates Increased Migrasome Formation by Cadaverine in RPE Cells
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease see attached file.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer’s comments: (Reviewer #1)
Overall, for a brief report, this study is interesting, and the data support the conclusions. However, the manuscript would benefit from improved clarity in writing. Therefore, this manuscript is recommended for publication after minor revisions.
Major points: 1. Line 17: The authors state that they produce a cell line overexpressing TSPAN4-GFP specifically in migrasomes. This sentence needs to be rephrased because TSPAN4 is expressed also in the cell body and retraction fibers.
Response 1: We appreciate to these helpful comments to improve our manuscript. According to the reviewer’s suggestion, we have revised the sentence.
- Line 21: In the abstract the authors state that the diamines increased the retraction fiber formation; however, no data supporting this claim are provided.
Response 2: According to the reviewer’s comment, we have revised the sentence by deleting the description.
- Line 47: The authors write that TSAPN4 localizes to retraction fibers through a mechanism involving TSPAN-enriched macrodomains. I believe this claim is inaccurate. It is more likely that TSPAN4 is recruited to the thin (and thus curved) retraction fibers due to its sensitivity to membrane curvature.
Response 3: According to the reviewer’s comment, we have revised it.
- The introduction lacks background information on TAAR8, PKA and their relationship to diamines. While some information about this is provided later in the manuscript, it should also be included in the introduction.
Response 4: According to the reviewer’s kind suggestion, we have further described about TAAR8 and PKA in the introduction part. But main text was described in the discussion part.
- In the abstract the authors write that they produce the RPE-TSPAN4-GFP cell line. However, in the methods section, they indicate that the cells were gifted by Prof. Li Yu, and in the acknowledgments, they mention that Prof. Li Yu provided the plasmids. Please clarify the correct origin of the cells and maintain consistency throughout.
Response 5: The GFP-msTSPAN4 plasmid was provided by Dr. Li Yu from China. However, we had generated a stable cell line using this plasmid in RPE cells in house. We have revised it to correct the description
- Line 93: Putrescine is not mentioned in the methods.
Response 6: According to the reviewer’s helpful comment, we have revised it.
- Line 116: It may be useful to include in a supplementary file a list of all the metabolites that were screened in their assay.
Response 7: According to the reviewer’s helpful comment, we added the list of metabolite library.
- The references to the figure panels are incorrect. For example: in line 120, they refer to Figure 1b instead of 1a, and in line 123, they refer to Figure 1C instead of 1B. Please carefully check all the references to the figures.
Response 8: We appreciate to your helpful comment, and we have revised it.
- Figure 1C: The authors show only one image for each condition. Could the authors provide more images in a supplementary file?
Response 9: According to the reviewer’s comment, we added some more pictures in supplementary figure S1.
- Line 136: The authors state that “cadaverine significantly inhibited PKA activation (Figure 1G). Moreover, compared with the control group, p-PKA levels decreased in cells treated with cadaverine or putrescine.” However, Figure 1G does not show a significant reduction in PKA levels; a significant reduction is shown only for p-PKA levels. can the authors clarify this statement?
Response 10: In the manuscript, we clarified that there was an increase in phosphor-PKA level, but not in total PKA level, consistent with the reviewer’s comment.
- Line 145- the authors mention that the cells were fixed before the imaging. Why were the cells fixed? Migrasomes can be imaged without fixation. Additionally, fixation is not mentioned in the methods section.
Response 11: According to the reviewer’s comment, we have revised the legend part.
- I did not see any supplementary movie file, so I cannot evaluate it
Response 12: According to the reviewer’s comment, we added movie files in supplementary Movie S1.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsCho et al. found putrescine can increase migrasome formation, and explained the relevant mechanism. The topic and innovation of this study align with the journal's requirements, and I am inclined to accept the manuscript upon revision.
The following suggestions for modification are provided.
1. All molecular formulas need to subscript numbers.
2. Could you please facilitate molecular docking experiments between putrescine and TAAR8?
3. The author needs to add the conclusion part at the end of the manuscript.
4. The author must meticulously examine the manuscript to ensure linguistic accuracy.
5. The reference format needs to be examined.
6. The iThenticate report shows that the repetition rate is relatively high and needs to be further reduced.
Comments on the Quality of English Languageneed minor revise
Author Response
Response to Reviewer’s comments: (Reviewer #2)
Reviewer #2: Cho et al. found putrescine can increase migrasome formation and explained the relevant mechanism. The topic and innovation of this study align with the journal's requirements, and I am inclined to accept the manuscript upon revision. The following suggestions for modification are provided.
- All molecular formulas need to subscript numbers.
Response 1: We appreciate to these helpful comments to improve our manuscript. According to the reviewer’s suggestion, we have revised the sentence.
- Could you please facilitate molecular docking experiments between putrescine and TAAR8?
Response 2: In a previous study, Izquierdo C et al. demonstrated the superposition of molecular docking of putrescine and cadaverine in TAAR8 molecular models (PLOS Computational Biology, 2018; https://doi.org/10.1371/journal.pcbi.1005945). We have already cited this paper as reference number 15.
- The author needs to add the conclusion part at the end of the manuscript.
Response 3: According to the reviewer’s comment, we have revised it by adding a conclusion part.
- The author must meticulously examine the manuscript to ensure linguistic accuracy.
Response 4: We appreciate to the comment to improve our manuscript. According to the reviewer’s suggestion, we have revised the manuscript.
- The reference format needs to be examined.
Response 5: We have revised the reference part to fit the format.
- The iThenticate report shows that the repetition rate is relatively high and needs to be further reduced.
Response 6: According to the reviewer’s suggestion, we have revised the manuscript to reduce prepetition rate.
Author Response File: Author Response.docx
