Extended Data Fig. 2: Heat shock and hyperosmotic shocks have similar phenotypes in yeast thermosensitive mutants.

During heat denaturation, protein stability (and so activity) decreases as the conformational entropy gain upon unfolding prevails over the entropy loss due to hydrophobic hydration⁵. Thermosensitive mutant proteins simply have lower melting temperatures than their wild type counterparts^92,93, i.e., they have lower intrinsic stability. In this study we highlight that, conceptually, from the perspective of water, a hypoosmotic treatment has similar thermodynamic consequences to increased temperature. Indeed, increasing temperature increases the ratio of free:structured water because the relative radius of hydration shells decreases and liberates water into the bulk solvent, which is equivalent to what happens under hypoosmotic conditions when water influx means that more bulk water is available compared with water in protein hydration shells. Thus, it is expected that a hypoosmotic treatment recapitulates the consequences of increased temperature for thermosensitive mutants: both treatments increase water availability and so reduce the relative cost of hydrophobic hydration for the solvent as a whole, leading to unfolding of a protein that is already on the threshold of denaturation. (a-f) Similarity of heat shock and hyperosmotic shock treatment for the S.cerevisiae mutant mss4^ts. (a-c) Validation of the mss4^ts mutant used in this study. (a) Principle of the experiment: S. cerevisiae cells expressing a PIP(4,5)P₂ GFP probe (PH-GFP) in a thermosensitive mutant background for the PIP(4) kinase mss4 are rapidly shifted from the permissible temperature (32 °C) to the restrictive temperature (39 °C). Inactivation of mss4 leads to loss of cortical PIP(4,5)P₂ and thus of cortical GFP signal. (b) PH-GFP signal over time (SDCM) upon rapid heat shock as depicted in (a). Elapsed time after temperature change in min. (c) Quantification of the effects seen in (b), see methods. Heat shock induces a quantitative loss of PH-GFP signal from the plasma membrane. (d-f) Hypoosmotic shock phenocopies heat shock for mss4^ts. (d) Principle of the experiment: S. cerevisiae cells expressing PH-GFP in a wildtype or mss4^ts mutant background were subjected to a hypoosmotic shock (405 mOsm l⁻¹ to 23 mOsm l⁻¹), then shifted back to isoosmotic medium and the cortical signal of PH-GFP was monitored by SDCM during the latter phase. This is the recovery time-course of the experiment presented in Fig. 1d–f. Experiment was performed at the permissive temperature (32 °C). (e) PH-GFP signal over time during recovery into isoosmotic medium as described in (a). Elapsed time after medium change indicated in min. (f) Quantification of the effects seen in (e). Note that on the contrary to WT, in the mss4^ts background, the PH-GFP signal is absent from the membrane in hypoosmotic medium, mimicking the heat shock treatment, but that this is alleviated upon shifting the cells into isoosmotic medium. (g-i) Similarity of heat shock and hyperosmotic shock treatment for the S. pombe mutant cut7-24. (g) Principle of the experiment: S. pombe cells expressing a spindle pole body probe (Sid4-GFP) and a microtubule probe (mCherry-Atb2, yeast homologue of α-tubulin) in a thermosensitive mutant background for the mitotic kinesin Cut7, cut7-24. Cut7 is a kinesin-5 motor, which is essential for the formation of a bipolar mitotic spindle by promoting the separation of the two spindle poles (yeast equivalent of centrosomes), and thus crucial for cell division^67,94. Consequently, temperature-induced degradation of Cut7 by shifting thermosensitive cut7-24 from the permissive (<36 °C) to the restrictive (>36 °C) temperature leads to the formation of monopolar spindles in almost all cells, while at the permissive temperature most cut7-24 mutant cells form bipolar spindles and proceed through cell division^18,67. (h) Representative field of views of cut7-24 cells expressing mCherry-Atb2 and Sid4-GFP in indicated conditions imagined by SDCM. (i) Quantification of the effect seen in (h) by manually scoring cells showing monopolar spindles in movies (see methods; Mean ± SD; n: number of fields of view (FOVs) quantified in 2–4 independent experiments pooled. Statistics unpaired t-test with respect to respective isoosmotic control. ****: P < 0.0001; N: total number of cells scored in each condition without averaging per FOV). Upon temperature shift to 37 °C almost 100% of mitotic cut7-24 cells form monopolar spindles that never reach bipolarity (mean ± SD 99 ± 1%, n = 17 FOVs), compared with 35 °C. As with S. cerevisae mss4^ts, exposing the cut7-24 cells at the permissive temperature to hypoosmotic condition (5% vol:vol of YES medium in water) mimicked the phenotype observed at the restrictive temperature and resulted in a dramatic increase of monopolar spindles. Moreover, the effects of the restrictive temperature were partially rescued by growing the cells overnight in medium supplemented with 50% D₂O. Note that hypoosmotic shock did not elicit any monopolar spindle in WT cells (of 422 cells, 0 cells formed monopolar spindles; data not shown). All panels in (b, e) correspond to single confocal planes while panels in (h) corresponds to maximum intensity projections (7 planes, Δz = 1 µm). Scale bars: 3 µm (b), 5 µm (e) and 10 µm (h).

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