Extended Data Fig. 4: Co-treatment with D₂O attenuates the effects of acute temperature and external osmolarity change, and attenuates cell death from heat shock.

(a-c) D₂O attenuates variation in osmotic potential as a function of PEG concentration and temperature. (a) since D₂O forms stronger H-bonds compared to H₂O, proteins should have a reduced impact on the structured/free water ratio in the presence of D₂O, and substitution of H₂O with D₂O should counter the effects of lowering temperature on the thickening of hydration shells by increasing the enthalpic favourability of hydration shells that incorporate heavy water. (b) Osmotic potential measured by vapour pressure for solutions of PEG-20kDa at indicated concentration in buffer containing 100% water (noted H₂O) or 50% heavy water/50% water (noted D₂O) solvent at indicated temperature. Note that the H₂O curves are identical to those presented in Extended Data Fig. 1, shown here for reference. Statistics: three-way ANOVA considering the solvent (D₂O vs. H₂O), PEG concentration and temperature as variables followed by a Tukey post-hoc test (significance indicated; ****: P < 0.0001, mean ± SEM). Note that the D₂O attenuates the effect of PEG concentration on osmotic potential, and that the effect of temperature is lower in D₂O versus H₂O. Both observations are consistent with increased enthalpic favourability of macromolecular-solvent interactions when H₂O is exchanged for D₂O. n = 3 independent osmometry curves measured for all conditions except D₂O/37 °C where n = 2; (c) Reduced sensitivity of \({I}_{{\rm{eff}}}^{s}\) to temperature for PEG-20kDa in D₂O (\({I}_{{\rm{eff}}}^{s}\) ± 95% confidence intervals are plotted). n: number of independent osmometry curves fitted simultaneously to evaluate \({I}_{{\rm{eff}}}^{s}\) in each condition. (d) The main consequence of a 47 °C heat shock on mammalian cells is the thermal denaturation of many different proteins. Thermally denatured proteins aggregate because they overload the cellular capacity to refold and degrade them; where aggregation is a second order process dependent on the concentration of denatured and partially unfolded proteins, and the failure to resolve this results in cell death^22,95. Protein unfolding occurs for two reasons: (1) macromolecules acquire sufficient kinetic energy to overcome the energy barrier for the entropic cost of hydrophobic hydration, and (2) because the relative cost of hydrophobic hydration falls as temperatures increase because the average number and strength of hydration bonds in bulk solvent is temperature dependent⁵. Both of these kinetic factors can therefore be understood in terms of solvent thermodynamics and so reducing water availability through increased solute concentration would be expected to increase protein thermal stability. However, for cells, such supraphysiological hyperosmotic treatment has two major consequences: the loss of cellular water²⁹, and an increased concentration of cellular macromolecules⁵⁰. Whilst the first should disfavour protein unfolding by lowering intracellular Ψ_π, the second will drastically favour the aggregation of proteins that have unfolded and so render cells at least as liable to cell death, if not more so. Similar to high sucrose concentrations, a stabilizing effect of D₂O on protein structure in solution is well established in the biochemistry field²⁴. The classical way to explain this effect is that D₂O forms stronger hydrogen bonds than H₂O (heavy ice melts at 3.8 °C). We therefore employed D₂O to demonstrate the solvent-dependence of cell death upon heat shock because D₂O immediately equilibrates over the cell membrane and so cannot affect cell volume. (e) Representative fields of view of adherent human foreskin fibroblasts cells, treated or not with a 45 min 50 °C heat shock in media containing the indicated percentages of D₂O (v/v), and stained with Calcein AM dye to assess viability. (f) Cell viability quantification in samples presented in (e). Mean ± SEM; n = 9 fields of view analysed per condition. Panel representative of N = 2 repeats. Statistics: two-way ANOVA followed by Dunnett’s post-hoc test (P value indicated). (g) D₂O attenuates protein condensation induced by acute hyperosmotic treatment. U2OS cells transiently expressing FusLC-GFP were equilibrated in media containing 0% or 50% D₂O for two min, then subjected to a 20 mOsm l⁻¹ hyperosmotic treatment in media containing 0% or 50% D₂O, respectively. The degree of FusLC-GFP was assessed by live SDCM before and after the treatment. (h) Log₂ of the fold change in granulosity index for nuclear FusLC-GFP upon osmotic challenge in the presence or absence of D₂O (median ± 95% confidence interval). Statistics: Mann-Whitney test. n: number of cells analysed. Note that condensation upon hyperosmotic shock is partially alleviated in the presence of D₂O. Scale bars: 10 µm (e), 200 µm (g).