Fig. 1: ASK3 forms macromolecular crowding-driven condensates under hyperosmotic stress.

a, b Subcellular localization of ASK3 5 min after osmotic stress in Venus-ASK3-stably expressing HEK293A (Venus-ASK3-HEK293A) cells. Hypoosmotic stress: ultrapure water-diluted medium, hyperosmotic stress: mannitol-supplemented medium, DIC: differential interference contrast, white bar: 20 μm, red bar: 2.5 μm. Data: center line = median; box limits = [Q₁, Q₃]; whiskers = [max(minimum value, Q₁ − 1.5 × IQR), min(maximum value, Q₃ + 1.5 × IQR)], where Q₁, Q₃ and IQR are the first quartile, the third quartile and the interquartile range, respectively; n = 12 (700 mOsm), 14 (600 mOsm), 15 (400 mOsm), 16 (150 mOsm, 225 mOsm, 300 mOsm and 500 mOsm) cells pooled from four independent experiments. Note that the signal intensity of DIC cannot be compared among the images. c Schematic diagram of a computational model for protein diffusion and clustering in a two-dimensional grid space. Red squares: ASK3 units, black squares: obstacles, pale yellow arrows: potential movements, blue squares: surface positions of the clusters. See Supplementary Note for the full description of the model. d, e A computational simulation for the relationship between the grid space and the number/size of ASK3 clusters. Results after 5 × 10⁶ steps in the rejection kinetic Monte Carlo (rKMC) method at each grid space are presented. Red shading: the assumed range corresponding to hyperosmotic stress (details in Supplementary Discussion), dashed line: the minimum of ASK3 clusters definition. Data: mean ± SEM, n = 18 simulations.