Theory of non-dilute binding and surface phase separation applied to membrane-binding proteins

Reviewer #1 (Public review):

Summary:

Biomolecular condensates are an essential part of cellular homeostatic regulation. In this manuscript, the authors develop a theoretical framework for the phase separation of membrane-bound proteins. They show the effect of non-dilute surface binding and phase separation on tight junction protein organization.

Strengths:

It is an important study, considering that the phase separation of membrane-bound molecules is taking the center stage of signaling, spanning from immune signaling to cell-cell adhesion. A theoretical framework will help biologists to quantitatively interpret their findings.

Weaknesses:

Understandably, the authors used one system to test their theory (ZO-1). However, to establish a theoretical framework, this is sufficient.

Reviewer #2 (Public review):

Summary:

The authors present a clear expansion of biophysical (thermodynamic) theory regarding the binding of proteins to membrane-bound receptors, accounting for higher local concentration effects of the protein. To partially test the expanded theory, the authors perform in vitro experiments on the binding of ZO1 proteins to Claudin2 C-terminal receptors anchored to a supported lipid bilayer, and capture the effects that surface phase separation of ZO1 has on its adsorption to the membrane.

Strengths:

(1) The derived theoretical framework is consistent and largely well-explained.

(2) The experimental and numerical methodologies are transparent.

(3) The comparison between the best parameterized non-dilute theory is in reasonable agreement with experiments.

Weaknesses:

(1) In the theoretical section, what has previously been known, compared to which equations are new, should be made more clear.

(2) Some assumptions in the model are made purely for convenience and without sufficient accompanying physical justification. E.g., the authors should justify, on physical grounds, why binding rate effects are/could be larger than the other fluxes.

(3) I feel that further mechanistic explanation as to why bulk phase separation widens the regime of surface phase separation is warranted.

(4) The major advantage of the non-dilute theory as compared with a best parameterized dilute (or homogenous) theory requires further clarification/evidence with respect to capturing the experimental data.

(5) Discrete (particle-based) molecular modelling could help to delineate the quantitative improvements that the non-dilute theory has over the previous state-of-the-art. Also, this could help test theoretical statements regarding the roles of bulk-phase separation, which were not explored experimentally.

(6) Discussion of the caveats and limitations of the theory and modelling is missing from the text.

Author Response:

We sincerely thank the reviewers and the editorial team for their thoughtful and constructive evaluation of our manuscript. We are very pleased that both reviewers and the Reviewing Editor found the work to be compelling and of interest to the community studying membrane-associated condensates. Below we outline our planned revisions in response to the public reviews.

Reviewer #1

We appreciate Reviewer #1’s positive evaluation of the study’s significance and the utility of our theoretical framework.

1. Understandably, the authors used one system to test their theory (ZO-1). However, to establish a theoretical framework, this is sufficient.

Response: We acknowledge this limitation. While we agree that additional systems would strengthen the generality of our theory, we note that the focus of this work is to introduce and validate a theoretical framework. As the reviewer notes, this is sufficient for establishing the framework. Nonetheless, we are open to further collaborations or future studies to test the model with other systems.

Reviewer #2

We are grateful for Reviewer #2’s detailed comments and will address each of the points as follows:

1. In the theoretical section, what has previously been known, compared to which equations are new, should be made more clear.

Response: We will revise the theory section to clearly distinguish previously established formulations from novel contributions.

1. Some assumptions in the model are made purely for convenience and without sufficient accompanying physical justification. E.g., the authors should justify, on physical grounds, why binding rate effects are/could be larger than the other fluxes.

Response: We will expand the discussion to provide key physical justification, especially to explain why binding rate effects are/could be larger than the other fluxes.

1. I feel that further mechanistic explanation as to why bulk phase separation widens the regime of surface phase separation is warranted.

Response: We will elaborate on the mechanism underlying this coupling.

1. The major advantage of the non-dilute theory as compared with a best parameterized dilute (or homogenous) theory requires further clarification/evidence with respect to capturing the experimental data.

Response: We will clarify this comparison more explicitly and highlight how the non-dilute model captures key nonlinear behaviors and concentration-dependent adsorption phenomena that the dilute model fails to reproduce.

1. Discrete (particle-based) molecular modelling could help to delineate the quantitative improvements that the non-dilute theory has over the previous state-of-the-art. Also, this could help test theoretical statements regarding the roles of bulk-phase separation, which were not explored experimentally.

Response: We appreciate the suggestion and agree that such modeling would be valuable. However, this is beyond the scope of the current study. We will add a discussion on how discrete simulations could be used to further test our theory in future work.

1. Discussion of the caveats and limitations of the theory and modelling is missing from the text.

Response: We will add a paragraph outlining caveats and limitations of the modelling.

We believe these changes will significantly improve the clarity and impact of our manuscript, and we thank the reviewers again for their valuable input.