Preprint reviews by Jonathan Humphries

The β3-integrin endothelial adhesome regulates microtubule dependent cell migration

Samuel J. Atkinson, Aleksander M. Gontarczyk, Tim S. Ellison, Robert T. Johnson, Benjamin M. Kirkup, Abdullah Alghamdi, Wesley J. Fowler, Bernardo C. Silva, Jochen J. Schneider, Katherine N. Weilbaecher, Mette M. Mogensen, Mark D. Bass, Dylan R. Edwards, Stephen D. Robinson

Review posted on 20th June 2017

Dear Authors,
Thanks for posting this very interesting collection of MS datasets and experimental observations that arose from your data. I have a number of suggestions and comments that I hope you feel will help with the analysis and presentation of the data. My comments mainly focus on the proteomics side of things.

1. The title is misleading as far as I can tell you have not defined the beta 3 integrin endothelial adhesome by MS-based proteomics. From the supplementary data files it is apparent that the adhesion complexes isolated comprise other integrins including a substantial amount of integrin a5b1. I understand that you provide additional evidence for a role of b3 integrin in MT stability but this equally could be due to the shift in balance between the proteins recruited to a5b1 and aVb3.
2. I think you are underselling the interest in what the FN-induced (a5b1 and aVb3) adhesome comprises in endothelial cells. You perform a nice subtractive proteomic approach to enable you to define the FN-enriched proteins (compared to PLL) but you don't tell us what these adhesome components are, or provide any comparison with other proteomic datasets or the Geiger literature-curated adhesome. This sort of analysis would help the field to understand the context-based composition of adhesion complexes (similarities and differences). I was left with the question 'what is the endothelial adhesome?'.
3. I was concerned by the approach you have taken to you proteomic analysis of the isolated complexes. Whilst on the face of it you have used good informatic tools (maxquant / perseus) I note that you performed the MS analysis from 3 pooled adhesion complex isolations. This will only permit a measure of the technical variability in the LC-MS/MS and not give you any idea of the variability in the biology i.e between adhesion complex isolations. Maybe there is a good statistical justification for this approach but it needs to be provided.
4. Also your MS dataset lists of proteins contain proteins from identifications with only 1 unique peptide. In my experience these '1-hit wonders' are a major source of variation in the quantitative values from MS outputs. Again please justify the inclusion of such identifications.
5. Please provide more details in the methods of the isolation of adhesion complexes and MS set up.
6. You have used one EC-derived cell type for all the experiments. Are your findings observed in other EC's such as non-transformed primary ECs?
7. In figure 1 it would be nice to see more IF staining of some cannonical adhesion proteins such as integrins (aV, a5, b3, b1) or paxillin / vinculin. You also note in the methods that you check the quality of the isolations by western blotting before MS analysis - could you provide any of this data to supplement the silver stains? It is reassuring to show blots for components you expect to see and don't expect to see in adhesion complexes before MS analysis.
8. Figure 2e why choose to blot for hspa1a? Are you saying this is an adhesion component? If the idea was to highlight equal recruitment to adhesion complexes why not blot for talin or vinculin?

I hope you find these comments useful. Thanks for sharing your data.
Jon Humphries
WTCCMR, The University of Manchester, UK

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