Consolidated peer review report (26 September 2024)
GENERAL ASSESSMENT
In this article, Kay refutes a major claim made by Watson et al., 2023. In the original publication, Watson et al. argue that macromolecular condensation acts as a cellular buffering mechanism to compensate for the effects of osmotic shock. In particular, they claim that, when water is drawn into or out of the cell due to hypo- or hyper-osmotic shock, respectively, macromolecular condensates rapidly capture (during hypo-osmotic shock) or release (during hyper-osmotic shock) free water to maintain a constant water potential (presumably in addition to a constant solute concentration and osmolality) within the cell. While Watson et al. find that macromolecular condensation in cells is responsive to osmotic shock, they do not measure intracellular water potential, osmolality, or macromolecular density in intact cells, and therefore do not directly demonstrate that biocondensation buffers any of these properties in living cells. In response, Kay argues that, while such a water buffer could temporarily maintain an osmolality differential across the membrane, this osmolality differential will necessarily drive water across the membrane until the osmolality within the cell equals the osmolality outside of the cell. Therefore, the steady-state behaviour is expected to be identical with and without the water buffer. Using the well-established pump-leak model for osmotic water transport, Kay further shows that the timescale at which a water buffer can maintain even a 10% osmolality differential across the membrane is at most a minute for a typical animal cell.
Overall, Kay 2024 provides a compelling rebuttal to a strong claim made by Watson et al. However, there is an opportunity for Kay to acknowledge nuanced situations where such a water buffering mechanism as that posited by Watson may be useful to cells. It’s also unclear if Kay has described a major inconsistency with Watson et al., particularly since the water release rate from condensates is not well quantified.
RECOMMENDATIONS
Essential revisions:
<ol>
<li>The author could acknowledge nuanced situations in which the water buffering mechanism described by Watson et al. may be useful to cells. For example, by slowing the rate of change of intracellular osmolarity due to osmotic shock and thus giving the cell time for more active feedback mechanisms to engage, or in buffering rapid fluctuations in extracellular osmolality</li>
<li>The flux of water across lipid membranes depends on the pressure difference across the membrane. The author simulated the situation with a 30 mOsm (~75 kPa) osmotic pressure difference. Considering that physiologically relevant pressure fluctuations can be much lower (a few kPa), is it possible that a water buffer would be more effective when there are small pressure differences across the cell membrane? The author should discuss this.</li>
<li>The author should cite the work from which they obtained the water buffer release rate.</li>
<li>It would be helpful to measure the dynamics of intracellular volume concurrently with biocondensate formation under cells exposed to osmotic shock (ideally under experimental conditions where cells either do or do not form condensates). If Watson et al.’s hypothesis is correct, the volume should not change (this seems unlikely). If your hypothesis is correct that buffering could only ever be temporary, one could then experimentally determine the buffering timescale by measuring the stall time between the shock and when the volume begins to change. The stall should also disappear in conditions where condensate formation is inhibited.</li>
<li>There are two inaccuracies in the discussion of membrane tension caused by osmotic pressure that would benefit from being corrected. First, when using Laplace’s Law to calculate membrane tension induced by 30 mOsm pressure, the author used a cell radius of 10 um and calculated a large (180 mN/m) membrane tension. This is significantly overestimated because the cell membrane can form local deformations via attachment to the cytoskeleton. These local deformations are typically around 10 - 100 nm, thus reducing the calculated membrane tension by 2-3 orders of magnitude, below the lysis tension of the membrane (1 - 10 mN/m). Second, the author is correct that measured resting membrane tension is low (&lt; 0.3 mN/m). However, recent evidence suggests that tension on the cell membrane can locally or transiently reach much higher levels (to &gt; 1mN/m). This is supported by activation of mechanosensitive ion channels such as Piezo1, which require an activation membrane tension ~ 1mN/m.</li>
<li>The discussion on non-equilibrium states is not very clear. Is the author suggesting that a water buffer can work more efficiently in an equilibrium system such as a giant vesicle?</li>
<li>Because the pump-leak model is generic, some contextual discussion of condensates would be helpful. For example, the dynamic formation of hydrogen bonds, van der Waals interactions, and possible charges resulting from hyperosmotic or low osmotic conditions that may indirectly participate in the hypothesis.</li>
<li>Has the author considered whether the thermodynamic driving forces associated with phase separation and condensate formation might affect the ability of condensates to buffer intracellular osmolality?</li>
</ol>
Optional suggestions:
<ol>
<li>The language of the article focuses on the role of membrane permeability, which is of course key. However, it might be helpful to explicitly state that an osmolality differential will always drive water across the membrane, so even if a water buffer could temporarily maintain such an osmolality differential, water will continue to flow across the membrane until the buffer is saturated and this differential is equalized.</li>
</ol>
REVIEWING TEAM
Reviewed by:
Rikki Garner, Postdoctoral Research Fellow, Harvard Medical School, USA: physics, biophysics, and physical/quantitative cell biology.
Tripta Bhatia, Assistant Professor, Indian Institute of Science Education and Research Mohali, India: soft matter, biological physics, membrane biophysics
Zheng Shi, Assistant Professor, Rutgers University-New Brunswick, USA: mechanics of biomolecular assemblies
Curated by:
Syma Khalid, Professor, University of Oxford, UK
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)

Macromolecular condensation is unlikely to buffer intracellular osmolality

Figures and data

Changes in osmolality and volume induced by an increase in extracellular osmolality in the presence or absence of a WB.