Fig. 3
Homotypic NPM1 droplets undergo reversible aging. a NPM1 concentrations measured within the dense (green, n ≥ 343 droplets) and light (black, n = 6) phases prepared in the presence of 5%, 15% and 30% PEG (see Methods). Values represent mean ± s.d. b Confocal microscopy images of NPM1-A488 droplets photobleached at t = 75 min (top row), 120 min (middle row), and 210 min (bottom row) after mixing 20 μM NPM1 with 5% PEG in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer; scale bar = 1 μm. c FRAP recovery curves of NPM1-A488 in 20 μM NPM1 droplets at t = 75 min (blue), 120 min (magenta), and 210 min (orange) after droplet formation in the presence of 5% PEG (left panel) and 15% PEG (right panel). Values represent mean ± s.d. for n ≥ 8 droplets. Under both crowding agent conditions, the fraction of NPM1 that recovered decreased at the later time points after mixing; ROI = 1 µm circular area in the center of the droplet. d Time-lapse fluorescence microscopy images of fusion between NPM1-A488 droplets formed 60 min (top row) or 180 min (bottom row) after mixing in the presence of 5% PEG. Droplets fused rapidly after incubation for 60 min, but fusion was very slow after incubation for 180 min. e An illustration of the experimental scheme used to monitor dissolution of aged NPM1 droplets formed in 5% PEG upon removal of the crowding agent. Light phase (90% of the solution) was gently removed and replaced with the same volume of buffer lacking PEG. Droplet dissolution was monitored over time using confocal fluorescence microscopy imaging. f Time-lapse imaging of aged droplets dissolving after removal of the crowding agent
