Extended Data Fig. 8: Condensation state of cellular marker as a function of osmotic challenge and changes in the global phosphorylation of the proteome.

(a,b) SH-SY5Y cells transiently expressing GFP or FusLC-GFP were subjected to indicated mild osmotic challenge and the condensation level of GFP or FusLC-GFP was quantified (granulosity index, see methods) upon hyperosmotic challenge (a) or hypoosmotic challenge (b). Mean ± SEM (n: number of independent cells analysed per condition). Data were normalized to the value of the granulosity before the osmotic challenge in each cell. Statistics: one sample t-test with a hypothetical mean value of 1 (P value as indicated). (c,d) Condensation of TIA1-GFP in U2OS cells upon osmotic challenge. (c) U2OS cells transiently expressing TIA1-GFP subjected to indicated osmotic challenge and imaged by SDCM (images correspond to maximum intensity projections of entire cell). Data were denoised using a wavelet “à trous” filter (see methods). Bottom panels correspond to zoom cropped views in the nucleus of different cells in the same conditions. (d) Variation of the granulosity index (mean ± SEM) of TIA1-GFP in the nucleus of U2OS cells upon osmotic challenge (see methods). Data were normalized to the value of the granulosity before the osmotic challenge in each cell. Statistics: one-way ANOVA with test for linear trend (P value as indicated). (e,f) Nucleolar condensation in response to a hypoosmotic challenge. (e) SH-SY5Y cells were stained with Nucleolar-ID for 15 min before being exposed to hypoosmotic shock (325 mOsm l⁻¹ to 162.5 mOsm l⁻¹) and imaged by SDCM. Elapsed time after hypoosmotic shock indicated in min. (f) Mean granulosity index over time (±SEM) in the condition described in (c). n: number of cells analysed. Granulosity was measured in the nucleus after deep-learning based nucleus segmentation (see methods). (g-h) FusLC-GFP condensation upon hyperosmotic shock is a passive process. (g) U2OS cells transiently expressing FusLC-GFP were treated (or not) with an established energy-depletion medium then subjected to a hyperosmotic shock (see methods). Blue arrows indicate spiky protrusions characteristic of energy depletion⁷⁷. (h) Granulosity index after the hyperosmotic shock normalized by its value in isosmotic conditions (mean ± SEM). Statistics, unpaired t-test, P = 0.7, n: number of cells analysed. FusLC-GFP condensation still occurs in energy-depleted cells. (i-k) FusLC-GFP condensation in response to global changes in protein phosphorylation. (i) SH-SY5Y cells transiently expressing FusLC-GFP were treated with 10 µM Staurosporine or 3 nM CalyculinA and FusLC-GFP condensation was monitored by SDCM at constant temperature and external osmolarity. Elapsed time after treatment indicated in min. Note the appearance of FusLC-GFP foci (arrows) upon global dephosphorylation by Staurosporin, and conversely, their disappearance upon global phosphorylation by CalyculinA. (j) Mean granulosity index over time (±SEM) in the condition described in (i). n: number of cells analysed. (k) SH-SY5Y cells transiently expressing FusLC-GFP were treated with Staurosporine, CalyculinA or DMSO vehicle for 50 min at 37 °C then FusLC-GFP was immunoprecipitated and analysed by GFP western blot after transfer from Phos-tag gels. Two technical replicates are shown. Note that FusLC-GFP runs at higher apparent molecular weight on the Phos-tag gel upon CalyculinA treatment, indicative of increased phosphorylation. Scale bars: 5 µm (c,e,g,i top panels); 1 µm (c,e,g,i bottom panels).