Fig. 4: Protein condensation in solution rapidly responds to acute changes of Ψ_π.

a, The condensation state of BSA–Alexa Fluor 647 (A647) (1 µM) and PEG–20kDa-rhodamine (1 mg ml⁻¹) in solutions in which free water availability was reduced by non-fluorescent PEG, assessed by SDCM (single confocal planes). Compared with BSA, PEG does not form condensates when the osmotic potential is low. b,c, Differential condensation of BSA and GFP as free-water availability decreases with increasing PEG concentration. BSA–A647 (1 µM) or GFP (1 µM) were imaged by SDCM (b) (27 °C) in PEG solutions of different osmotic potentials (Extended Data Fig. 1) and automatically quantified (c). For c, data are mean ± s.e.m. of the condensation ratio (Methods). Statistical analysis was performed using two-way ANOVA with Šídák’s test comparing BSA versus GFP; P values are indicated. The n values indicate the number of images per sample (Extended Data Fig. 9e). d,e, BSA condensation is not due to simple crowding effects. BSA–A647 (100 nM) was incubated at 27 °C in solutions of PEG (300 mg ml⁻¹) or dextran (350 mg ml⁻¹) of similar size, with the condensation state imaged by SDCM (d) and quantified (e). For e, data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA followed by Šídák’s test comparing PEG versus dextran; P values are indicated. n values indicate the number of images per sample. Dextran increases molecular crowding without causing BSA to form condensates, in contrast to PEG, which lowers free-water availability significantly more than dextran (Extended Data Fig. 1). f, Plot of \({I}_{{\rm{eff}}}^{s}\) (±95% confidence interval) versus temperature for PEG-10kDa. The n values indicate the number of independent osmometry curves fitted simultaneously to evaluate \({I}_{{\rm{eff}}}^{s}\) in each condition. PEG–solvent interactions increase as the temperature decreases (Extended Data Fig. 1). g,h, The effect of temperature on BSA condensation. BSA–A647 (100 nM) in PEG-10kDa (250 mg ml⁻¹) at the indicated temperatures was imaged using SDCM (g) and the condensation ratio was calculated (h). For h, data are mean ± s.e.m. Statistical analysis was performed using Mann–Whitney U-tests. The n values indicate the number of images analysed. For a, b, d and g, scale bars, 5 µm.

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