Probing the force-from-lipid mechanism with synthetic polymers

Reviewer #1 (Public review):

Summary:

The authors study the effect of the addition of synthetic amphiphile on the gating mechanisms of the mechano-sensitive channel MscL. They observe that the amphiphile reduces the membrane stretching and bending modulii, and increases the channel activation pressure. They then conclude that gating is sensitive to these two membrane parameters. This is explained by the effect of the amphiphile on the so-called membrane interfacial tension.

Strengths:

The major strength is that the authors found a way to tune the membrane's mechanical properties in a controlled manner, and find a progressive change of the suction pressure at which MscL gates. If analysed thoroughly, these results could give valuable information.

Weaknesses:

The weakness is the analysis and the discussion. I would like to have answers to some basic questions.

(1) The explanation of the phenomenon involves a difference between interfacial tension and tension, without the difference between these being precisely defined. In the caption of Figure 4, one can read "Under tension, the PEO groups adsorb to the bilayer, suggesting adsorption is a thermodynamically favorable process that lowers the interfacial tension." What does this mean? Under what tension is the interfacial tension lowered? The fact that the system's free energy could be lowered by putting it under mechanical tension would result in a thermodynamic unstable situation. Is this what the authors mean?

(2) From what I understand, a channel would feel the tension exerted by the membrane at its periphery, which is what I would call membrane tension. The fact that polymers may reorganise under membrane stretch to lower the system's free energy would certainly affect the membrane stretching modulus (as measured Figure 2E), but what the channel cares about is the tension (I would say). If the membrane is softer, a larger pipette pressure is required to reach the same level of tension, so it is not surprising that a given channel requires a larger activation pressure in softer membranes. To me, this doesn't mean that the channel feels the membrane stiffness, but rather that a given pressure leads to different tensions (which is what the channel feels) for different stiffnesses.

(3) In order to support the authors' claim, the micropipette suction pressure should be appropriately translated into a membrane tension. One would then see whether the gating tension is affected by the presence of amphiphiles. In the micropipette setup used here, one can derive a relationship between pressure and tension, that involves the shape of the membrane. This relationship is simple (tension=pressure difference times pipette radius divided by 2) only in the limit where the membrane tongue inside the pipette ends with a hemisphere of constant radius independent of the pressure, and the pipette radius is much smaller than the GUV radius. None of these conditions seem to hold in Figure 2C. On the other hand, the authors do report absolute values of tension in the y-axis of Figure 2D. It seems quite straightforward to plot the activation tension (rather than pressure) as a function of the amphiphile volume fraction in Figure 2B. This is what needs to be shown.

(4) The discussion needs to be improved. I could not find a convincing explanation of the role of interfacial tension in the discussion. The equation (p.14) distinguishes three contributions, which I understand to be (i) an elastic membrane deformation such as hydrophobic mismatch or other short-range effects, (ii) the protein conformation energy, and (iii) the work done by membrane tension. Apparently, the latter is where the effect is (which I agree with), but how this consideration leads to a gating energy difference (between lipid only and modified membrane) proportional to the interfacial tension is completely obscure (if not wrong).

(5) I am rather surprised at the very small values of stretching and bending modulii found under high-volume fraction. These quantities are obtained by fitting the stress-strain relationship (Figure 2D). Such a plot should be shown for all amphiphile volume fraction, so one can assess the quality of the fits.

Reviewer #2 (Public review):

Summary:

The manuscript describes how synthetic polymers, primarily poloxamers of different sizes, influence bacterial mechanosensitive channel MscL gating by modifying the interfacial tension of the membrane. The authors expressed MscL in U2OS cells and chemically blebbed the cells to derive giant plasma membrane vesicles (GPMVs) containing MscL G22S. They applied micropipette aspiration on GPMVs to obtain bending rigidity (kc) and area expansion modulus (kA) and used patch clamping to obtain activation pressure. They found a negative correlation between kc and kA with activation pressure and attributed the changes to activation pressure to the lowering of the interfacial tension in the presence of polymers. They carried out coarse-grain molecular dynamics simulations and showed that under tension the hydrophilic PEO group adsorbs to the bilayer more, thereby lowering the interfacial tension. Besides MscL, they showed similar results with TREK-1 activation. The conclusion that differences in interfacial tension are what drive the changes in activation pressure is based on using a thermodynamic model.

Strengths:

(1) Reveals that synthetic polymer that lowers bending rigidity and area expansion modulus increases activation pressure of mechanosensitive channel by lowering interfacial tension - this is an important finding.

(2) General data quality is high with detailed and thorough analysis. The use of both micropipette aspiration and patch clamp in the same study is noteworthy.

(3) Discussion on nanoplastics and their effect on membrane properties and therefore their impact on mechanosensitivity is interesting.

Weaknesses:

Interfacial tension is not experimentally measured. Given the main argument of this paper is that synthetic polymers reduce interfacial tension, which increases MS channel activation pressure, it would be prudent to show experimental measurements to bolster their analysis.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors set out to test the "force from lipids" mechanism of mechanosensitive channel gating, which posits that mechanical properties of the membrane are directly responsible for converting membrane tension into useful energy for channel gating. They employ amphiphilic polymers called poloxamers to alter membrane mechanical properties and relate those to the threshold of mechanical activation of the MscL channel of E.coli.

The authors heterologously express the channel, perform electrical recordings, and assess the mechanical properties of vesicles derived from the same membranes. This allows them to directly compare derived mechanical parameters to channel gating in the same environment.

They further repeat experiments in an eukaryotic mechano-channel and show that the same principles apply to gating in this very different protein, providing support for the force from lipids hypothesis.

Strengths:

In this work, characterization of the mechanical properties of the plasma membrane and electrical recordings of channel activity are carried out in membranes derived from the same cells. This is a nice contribution to these experiments since usually these two properties are measured in separate membranes with differing compositions. The experiments are of high quality and the data analysis and interpretation are careful.

Weaknesses:

It is not clear to this reviewer what the relationship is between the mechanical properties the authors measure, the membrane area expansion modulus, and bending rigidity, to what they call "interfacial tension".

Author response:

We appreciate the time and thoughtful reviews of all 3 reviewers. Ahead of a full revision of the paper, we would like to address a couple of points the reviewers have raised that we plan to address in more detail in our full revision.

(1) The relationship between membrane tension and interfacial tension: The major request by reviewers was for a better explanation of the relationship between measured mechanical parameters and membrane interfacial tension. We plan to include a schematic of the different forces at play in the membrane and to clarify our discussion and here, provide a brief explanation.

In our study, we identified a relationship between channel activation pressure and two membrane mechanical properties (area expansion modulus (K_A) and bending rigidity (K_c)) though we did not find a correlation between channel activation pressure and a third mechanical property (membrane fluidity). Through further computational analysis of the membranes, we identified an additional property called interfacial tension that helps unify and explain our results. Interfacial tension (γ) is a property akin to surface tension that reflects the chemical composition at the interface of the membrane (between the polar headgroups of the lipids and the hydrophobic acyl chains of the lipids) and balances the repulsive interaction of the nonpolar hydrocarbon chains with the polar headgroup regions of the lipids. In the established polymer brush model, the expansion modulus is proportional to the interfacial tension (W. Rawicz, Biophyiscal Journal, 2000)

γ = K_A/C,

where C is a constant. Interfacial tension occurs at the boundary between the lipid bilayer and external aqueous environment and is different from mechanical tension. While mechanical membrane tension (t) reflects a physical force in plane with the membrane, interfacial tension reflects the chemical composition at each interface of the membrane. While mechanical membrane tension depends on the size and shape of the membrane, interfacial tension is independent of these features and depends on the molecular composition of the liquid-liquid interface. An expanded discussion on this topic was recently provided (Lipowsky. Faraday Discussions. 2024). While distinct, these two properties can be related to one another via the area expansion modulus (K_A). Typically, one would imagine that upon reducing interfacial tension, and correspondingly reducing the K_A, it should now take less energy to stretch the membrane to the same extent and should reduce the activation pressure (and corresponding in plane mechanical tension ) required to open an embedded mechanosensitive channel. Interestingly though, interfacial tension also works to pull the channel open so that a reduction in interfacial tension also means more energy will be required to open the channel. We find that reductions in interfacial tension and corresponding increased energy required to open embedded channels outweighs the reduced tension that should be required to stretch the membrane. We plan to more clearly explain this tradeoff in our revision. Overal, our findings identify the exact properties driving mechanosensitive channel behavior in our study. Further, they provide a guide to understanding how and why shifts in mechanosensitive channel activation occur by connecting chemical composition changes to the changes in membrane tension propagation in a given membrane.

(2) Data presentation to support determined area expansion modulus and bending rigidity values: We will show stress strain curves used to derive Ka and kc values

(3) Address why membrane tension data was not shown for ephys experiments: The micropipette and patch clamp setups are different, and we did not use the same system for both measurements. In fact, limitations in tools that would allow for concurrent tension measurements while conducting channel activation measurements have limited our understanding of the role of membrane tension on mechanosensation to date. While recent studies have attempted to resolve this limitation through the design of new tools that enable concurrent monitoring of mechanosensitive channel activation and membrane tension (Lüchtefeld et al. Nature Methods. 2024), these tools were not available to us during our study or now. Because our study also attempted to connect these two features (membrane tension and channel activation) but we lacked tools to do so simultaneously, we used two sets of measurements to separately uncover membrane mechanical properties and channel activation pressure.

One reason it is difficult to measure membrane tension during a typical patch clamp study is because of limitations in the imaging equipment and pipettes used for this assay. The experiment is usually done by looking through the eyepiece and the pipette angle is around 45 degrees from the plane of the stage so it would be hard to visualize changes in the patch geometry in the tip of the pipette. Basically, we are able to see the pipette touch the GMPV, but cannot resolve the patch moving up the pipette. In response to the reviewer comment that tension=pressure difference times pipette radius divided by 2, we were unable to measure the radius and changes in radius of a patch upon increases in applied pressure due to the above mentioned imaging constraints. This limitation is why we were unable to directly measure applied tension with our current patch clamp set up.

(4) Interfacial tension is not experimentally measured: Interfacial tension = K_A /C where C is a constant (typically C=4 for bilayer membranes). The best way to measure interfacial tension is to determine K_A (the area expansion modulus), which we have experimentally done by generating stress vs strain curves for GPMVs. In literature, reductions in interfacial tension of a membrane are typically experimentally determined by measuring a corresponding reduction in the associated K_A value (eg. Ly and Longo. Biophys J. 2004). We have similarly followed this approach.