Fig. 4: Substitution of p14^ARF hydrophobic residues blocks p14^ARF meso-scale ordering and restores NPM1 mobility within condensates.

A p14^ARF structural features, including PSI-PRED4.0 secondary structure prediction (2^o Struc.), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.). The CIDER analysis for p14^ARFΔH1-3 is shown below. B Zoomed in regions from confocal fluorescence micrographs of NPM1-AF488 in condensates with p14^ARF (top) and p14^ARFΔH1-3 (bottom). Scale bars = 10 µm. C Index of dispersion for NPM1 in condensates with p14^ARF (gray boxes, whiskers and trace; derived from n = 6, 6, 6, 7, 6, 6, 7, 6, 6, 6, 7 images) and p14^ARFΔH1-3 (blue boxes and whiskers and trace, where n = 5, 4, 6, 6, 8, 6, 6, 6, 6, 6, 6 images). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The black arrow highlights the increased NPM1 saturation concentration, ΔC_sat, upon substitution of p14^ARF hydrophobic residues to Gly/Ser spacer residues. The gray arrow highlights the reentrant phase transition, which occurs at elevated p14^ARF concentrations. D ΔG^tr for NPM1 in condensates with p14^ARF (gray boxes, whiskers and trace, where n = 696, 42, 61, 159, 225, 285, 333, 276, 306, 227, 773 condensates) and p14^ARFΔH1-3 (blue boxes, whiskers and trace, where n = 2561, 1787, 29, 31, 82, 92, 134, 153, 166, 145, 162 condensates). Whiskers extend from the box to the furthest point within 1.5x the inter-quartile range. The C_sat for NPM1 increases when p14^ARF hydrophobic residues are substituted. The gray arrow highlights the destabilization of NPM1 during the reentrant phase transition. E CV-SANS curves for p14^ARFΔH1-3-[²H]-NPM1 condensates collected at 50% D₂O, where p14^ARFΔH1-3 is contrast matched ([²H]-NPM1 detected; green trace), at 85% D₂O where [²H]-NPM1 is contrast matched (p14^ARFΔH1-3 detected; blue trace), and p14^ARFΔH1-3-NPM1 condensate at 100% D₂O for full scattering intensity (NPM1 and p14^ARFΔH1-3 detected; gray trace). All curves are offset for clarity. Scatter points represent the average, the error bars represent the uncertainty derived from the counting statistics of the SANS instrument, as described and cited in the Methods. F Schematic describing condensed NPM1 with extended IDR conformations. G Schematic describing condensed p14^ARFΔH1-3 in an extended conformation. H FRAP of NPM1-AF488 within condensates shows that substitution of p14^ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. Scale bars = 1 µm. I FRAP recovery curves for p14^ARF-NPM1 and p14^ARFΔH1-3-NPM1 condensates with fits, as described in Methods (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 curves for each condition, the p-value is shown in the figure). J NPM1-AF488 D_App values extracted from the FRAP recovery curves in panel I (statistical significance was assessed by two-sided Wilcoxon rank-sum test, n = 10 D_App values for each condition, the p-value is shown in the figure).