Fig. 4: Architecture of the nucleolus arises through multiphase liquid miscibility and immiscibility of DDX18 with NPM1 and FBL, respectively.

a, b Biophysical properties of the mixtures of purified DDX18-GFP (20 μM) and NPM1-RFP (10 μM) recombinant proteins in vitro in a buffer comprised of 150 mM NaCl, 25 mM Tris-HCl, pH 8.0. Adding NPM1-RFP solution without PEG (i.e., no droplets formed) to the preformed DDX18 (a) established droplets with “core-shell” like structure, imaged with RFP, GFP, and merging RFP/GFP (b). The experiment was repeated independently twice with similar results. c Biophysical properties of the mixtures of purified DDX18-GFP (20 μM), NPM1-CFP (10 μM), and RFP-FBL (20 μM) with or without rRNA/snoRNA in a buffer comprised of 150 mM NaCl, 25 mM Tris-HCl, pH 8.0, detected by GFP, CFP, and RFP, respectively. Merged images and quantification of the formation of indicated core-shell structures are shown, where the percentages reflect the proportion of fields of view exhibiting the core-shell structures. The experiment was repeated independently twice with similar results. d Images of normal (shLuc) and disrupted (shDDX18#1) nucleolar condensate organization. FBL labels the dense fibrillar component (DFC); NPM1 labels the granular component (GC). The experiment was repeated independently twice with similar results. e Quantification of immunostaining images from control and KD cells (shDDX18#1 and shDDX18#3) showing the percentage of inclusive DFC localized cells (represented by the top part of panel d) or exclusive DFC localized cells (represented by the bottom part of (d). Quantifications were performed using 152 cells from shLuc, 211 cells from shDDX18#1, and 202 cells from shDDX18#3, based on three technical replicate cultures. Data are presented as mean values \(\pm\) SD. f A proposed model showing that DDX18 coordinates with NPM1 to safeguard nucleolus organization. (Created in BioRender. Malik, V. (2025) https://BioRender.com/v27l155). Source data are provided as a Source Data file.