Model Membrane Systems Used to Study Plasma Membrane Lipid Asymmetry

Round 1

Reviewer 1 Report

The authors present a well-written manuscript, in which they comprehensively review the most recent technical and experimental advances in the generation of asymmetric artificial/model membranes, including state-of-the art analytical approaches to quantify membrane asymmetry and membrane rigidity. An additional chapter is dedicated to the use of molecular dynamics simulations as an indispensable technique in the field, useful to estimate physicochemical parameters related to leaflet tension and of relevance to extend and complement current models of membrane asymmetry.  

Main focus of the manuscript is certainly the description of chemical-assisted and enzyme-based strategies underlying the preparation of asymmetric model membranes, thereby covering also few examples of protein integration into membranes. In my view, the current manuscript is of importance for both, specialists and non-specialists in the field. It gives a limited but sufficient overview on the methodological concepts of the in vitro preparations of asymmetric membranes of defined size, lipid composition, as well as their analyses, while on the other hand it imbeds the concepts into some interesting biologically and biophysically relevant questions. I feel that the manuscript in its present form, including the cited references, provides solid information to scientists who aim to obtain insights into current techniques of asymmetric membrane preparations and those engaged in applying or improving the methodological framework for addressing fundamentals of asymmetric membrane function in biological processes. I fully recommend publication of the manuscript.

Author Response

We thank the reviewer for their kind comments.

Reviewer 2 Report

The authors have summarized and effectively described past results regarding experimental and theoretical approaches to various membrane systems. The contents of the symmetry of the membrane are also consistent with the aim of this journal, so this review paper is considered worthy of publication.

However, we wish there were a few very simple questions and corrections as following.

• There are 7 authors in this paper. Oddly enough, all authors are marked as corresponding authors. Is this possible? If possible, we think it is good to briefly express the contributions of the authors in this paper.
• Due to the nature of the review paper, it is essential to extract pictures from various papers. As a result, the form of the graph is different. If it is possible to express in a unified form, we would like to recommend correction.
• The authors added various reference papers to describe, but it seems that the papers within the last 3 years are relatively lacking. It is recommended to add the most recent results as it will be of great help to reflect the latest trends.
• Lastly, the format of references is not unified. Please write in one unified form, such as an abbreviation or full name.

We strongly recommend that you publish any corrections to these parts.

Author Response

However, we wish there were a few very simple questions and corrections as following.

• There are 7 authors in this paper. Oddly enough, all authors are marked as corresponding authors. Is this possible? If possible, we think it is good to briefly express the contributions of the authors in this paper.

H.L.S., K.B.K, T.A.E., M.D., J.J.K., F.A.H., and J.K. wrote the manuscript, designed the figures, and revised the manuscript.

Author contributions have been added to the manuscript.

• Due to the nature of the review paper, it is essential to extract pictures from various papers. As a result, the form of the graph is different. If it is possible to express in a unified form, we would like to recommend correction.

We thank the reviewer for his/her  comment. Figures, particularly 1, 3, 7, and 8 have been revised, addressing the reviewer’s comment.

• The authors added various reference papers to describe, but it seems that the papers within the last 3 years are relatively lacking. It is recommended to add the most recent results as it will be of great help to reflect the latest trends.

We have addressed this comment by including and discussing the papers by Dokotorova et. al. 2019,  Nguyen et. al. 2019, and Scott et. al. 2019.

• Lastly, the format of references is not unified. Please write in one unified form, such as an abbreviation or full name.

This has been addressed in the revised version of the manuscript.

• We strongly recommend that you publish any corrections to these parts.

We thank the reviewer.

Reviewer 3 Report

The work by Scott et al, reviewed progress on preparing and understanding asymmetric lipid membranes that, in some cases, have shown different behaviors compared with the symmetric counterpart. Overall, the paper covers mainstream methods and key concepts, provides easy-to-follow illustrations that are accessible to general audience, and cites important as well as up-to-date references. A few minor comments are suggested below:

1. Page 5 and Figure 1. The main text talked about effects of vesicle diameter and referred to Fig. 1B, which should be Fig. 1D. Same for the discussion on temperature and composition effects.
2. Figure 7. The figure shows solid blue lines and referred to as red line, even though the original paper indeed used red lines. The authors might want to fix this.
3. The MD simulations. Is the challenges to calibrate the force fields unique to asymmetric lipid bilayers? Or does it apply to the symmetric case as well? The two major challenges seem equally important to the MD simulation of symmetric case.

Author Response

The work by Scott et al, reviewed progress on preparing and understanding asymmetric lipid membranes that, in some cases, have shown different behaviors compared with the symmetric counterpart. Overall, the paper covers mainstream methods and key concepts, provides easy-to-follow illustrations that are accessible to general audience, and cites important as well as up-to-date references. A few minor comments are suggested below:

1. Page 5 and Figure 1. The main text talked about effects of vesicle diameter and referred to Fig. 1B, which should be Fig. 1D. Same for the discussion on temperature and composition effects.

We have addressed issue this in the revised version of the manuscript.

2. Figure 7. The figure shows solid blue lines and referred to as red line, even though the original paper indeed used red lines. The authors might want to fix this.

The word red has been replaced with blue to ensure the text and image match.

3. The MD simulations. Is the challenges to calibrate the force fields unique to asymmetric lipid bilayers? Or does it apply to the symmetric case as well? The two major challenges seem equally important to the MD simulation of symmetric case.

The challenges related to the experimental calibration/validation of MD simulations of asymmetric bilayers are indeed unique to membranes that are compositionally asymmetric. The reason they don’t apply to symmetric bilayers is the fact that the latter have the same numbers and types of lipids in their two leaflets and as a result, the leaflets are stress-free. Similarly, experimental symmetric model membranes are well defined, and their properties have been historically used to develop and refine lipid force field parameters. The challenges described in the manuscript preclude such direct crosstalk between simulation and experiment for asymmetric membranes. We have edited the text in an attempt to clarify this point.  

We thank the reviewer for their kind comments concerning our manuscript and for the helpful corrections that will improve this manuscript.

Reviewer 4 Report

The authors have summarized current methods and recent advances in the in vitro generation of asymmetric lipid vesicles, as well as efforts for their in silico simulation and reconciliation of computational vs. experimental results. The authors give a comprehensive review of these methods and highlight their potential advantages, focusing on leaflet coupling and the physical properties of the lipid bilayer.

Overall, the review is well-written and easy to follow, the illustrations are useful and adequate. I do not have significant changes to suggest for the manuscript.

Minor practical issues should be attended:
- figure captions in general refer to "red lines", but those lines were blue in the graphs.
- page 16 needs a dash in the phrase "between 1050 micron".
- spell checking should be run to correct minor errors.

Author Response

Minor practical issues should be attended:
- figure captions in general refer to "red lines", but those lines were blue in the graphs.

The word red has been replaced with blue to ensure the text and image match.

- page 16 needs a dash in the phrase "between 1050 micron".

A dash has been added.

- spell checking should be run to correct minor errors.

Spell check has been run and errors corrected.

We thank the reviewer for their kind comments concerning our manuscript and for the helpful corrections that will improve this manuscript.

Reviewer 5 Report

The cell biophysics of is very important for the biomedical science and its wide range applications such as the following of the interactions processes between cells. The investigation of lipid-lipid and lipid-protein interactions on asymmetric model membranes using many different methods for their preparation as well as computational techniques is interesting topic for readership of the Symmetry.

 In this work, authors reviewed sufficiently developed techniques in studies of PM asymmetry that have potential to answer to many crucial questions associated with membrane asymmetry as an important membrane feature.

However, minor revision is required as given below:

1. In page 18 of 39: " Using the phase transfer method, Elani et al. prepared POPC aGUVs with their inner and outer leaflets labeled with NBD-PE and Rh-PE ", authors should add the text with "..Rh-PE ( 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl) (ammonium salt). "

2. In page 25 of 39, authors should correct the next sentence: " Leaflet tension is zero both in symmetric bilayers and in asymmetric bilayers with appropriately balanced numbers of lipids in the two leaflets (Fig. 11A)⁹⁶." with " leaflets (Fig. 11 Left)⁹⁶".
3. In page 25 of 39, "A non-zero leaflet tension, also termed differential stress, can change the physical properties of the bilayer, including its structure and elastic constants, in a magnitude-dependent manner (Fig. 11B).^71,96" should be replaced with …. manner (Fig. 11 Right).^71,96".

Author Response

However, minor revision is required as given below:

1. In page 18 of 39: " Using the phase transfer method, Elani et al. prepared POPC aGUVs with their inner and outer leaflets labeled with NBD-PE and Rh-PE ", authors should add the text with "..Rh-PE ( 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl) (ammonium salt). "

The text has been added.

2. In page 25 of 39, authors should correct the next sentence: " Leaflet tension is zero both in symmetric bilayers and in asymmetric bilayers with appropriately balanced numbers of lipids in the two leaflets (Fig. 11A)⁹⁶." with " leaflets (Fig. 11 Left)⁹⁶".

The correction has been made.

3. In page 25 of 39, "A non-zero leaflet tension, also termed differential stress, can change the physical properties of the bilayer, including its structure and elastic constants, in a magnitude-dependent manner (Fig. 11B).^71,96" should be replaced with …. manner (Fig. 11 Right).^71,96".

The correction has been made.

We thank the reviewer for their kind comments concerning our manuscript and for the helpful corrections that will improve this manuscript.