Peer-reviewed by 2 reviewers with median rating of 13/20. Review process was triple-blinded.
Round 1 (13/20)
Round 2 (13/20)
Conceptual advance and Impact4.5
The only major problem are all the references to "clusters" in the title, abstract, conclusion, etc. The problem is that there is absolutely no evidence for any clusters. If all these references are removed and a few small issues fixed, then this paper is acceptable for Matters.
There are no data about 'clusters' in this manuscript. All references to "clusters" must be removed from the title, abstract, and conclusion.
The sentence about ADAM10 is unnecessary for an abstract. If this is a prevailing theory in the field, add it to the introduction.
Remove all references to 'clusters'.
Fig5A-B and Fig6 have no error bars. I’m not sure the editorial policy of the journal, but single measurements without repeats are not usually acceptable as representative of a real observation.
What is on the x-axis of Fig7A.
reference to clusters here is uncited. is this conjecture?
No evidence for clusters.
Results & Discussion
Fig2 (bottom) is cited for two different experiments. The statement referencing kinetics of channel formation doesn’t make sense with the data shown.
Insensitivity of unpolarized MDCKs is a crucial result, and particularly surprising given that 1day filter-grown are highly sensitive. If unpolarized cells are really insensitive, this data are important to show.
MBCD may cause leakage of K+ by itself. It is inappropriate to show the effect of the toxin as % of initial. Rather, it should be shown as % of untreated control.
Do all experiments use 15nM toxin? Or 500ng/ml? Please put into the same units and specify which concentrations were actually used.
I think this section is potentially very useful in that it forces the authors to consider the limitations of their experiments. What the authors have included is disappointing. There are several limitations of this work that should be pointed out. Beyond the ones listed above:
1 - all pore formation results measure K+ only. Its possible that some pores are formed that dont leak K+ or that there is an active K+ efflux induced by toxin.
2 - MBCD treatment is extremely pleitropic and its difficult to pin any effect directly on the lack of cholesterol and not the many possible downstream responses of the membranes and the cells.
3 - there seems to be an order of magnitude difference in sensititivity of cells to the toxin (MBCD vs HeLa vs MEB4). Why?
If 'clusters' were to be included anywhere in the manuscript, it should be here.
Conceptual advance and Impact8
I suggest "phosphatidylcholine clusters" instead of "phospho choline head groups clusters". The toxin does not recognizes sphingomyelin, and the hydrophobic part of PC might be relevant in the presentation of the anti get, thus the current title is misleading to me
In the abstract and the text, alpha should be spelt out or rendered with the Greek alpha, not with a.
Results & Discussion
A part from glyco lipids, is the lipid composition of the two cell lines MEB4 and GM95 comparable in terms of cholesterol and phospholipid content? If not it would be more stringent to compare the behavior of MEB4 in the presence or absence of a glucosylceramide synthase inhibitor such as PDMP.