Fig. 1: Phase separation of Nup98 FG domains shows a lower critical solution temperature (LCST) behaviour.
From: Atomic resolution dynamics of cohesive interactions in phase-separated Nup98 FG domains
a Both the wild-type Mac98A FG domain and an engineered perfectly repetitive sequence, prf.GLFG52 × 12, phase separate to near-spherical particles following purification and dilution out of 2 M guanidine hydrochloride with an aqueous buffer (Methods), as shown by microscopy (top: phase-contrast microscopy images, below: confocal laser scanning microscopy images). Both FG phases exhibit NPC-like transport selectivity: they allow the partition of a nuclear transport receptor NTF2 (labelled with Alexa Fluor 488, green) to a partition coefficient of ~2000, but exclude the inert probe mCherry protein (partition coefficient < 0.05) (red). b Sequence of prf.GLFG52 × 12 containing 52 perfect repeats of a 12-mer peptide. c A dilution of prf.GLFG52 × 12 (15 μM at 400 mM NaCl) at 4 °C does not display phase separation, however when warmed up to 37 °C, it turns turbid readily, indicating phase separation. The process is reversible by cooling back to 4 °C. d 20 μM dilutions of prf.GLFG52 × 12 prepared at room temperature and centrifuged at different temperatures. SDS samples of the obtained pellets (FG phase) and supernatants (aqueous phase) are shown on SDS-PAGE, followed by Coomassie blue staining. Critical concentrations for individual conditions are taken as the concentrations of the supernatants. Full scans of gels with molecular weight markers are provided in the Source Data file. *At 7 °C, 100 μM of prf.GLFG52 × 12 is required to determine the critical concentration. e A dilution of prf.GLFG52 × 12 analysed by Dynamic Light Scattering (DLS), with the temperature increased from 2 to 40 °C (red). A sharp increase in hydrodynamic radii (from ~5 nm, expected for monomers26,34, to final radii of ~1000 nm, expected for the multimeric FG particles) indicates phase separation at a transition temperature (Tp) ~16 °C. As in c, the phase separation is reversible by cooling (blue). f DLS analysis repeated with different concentrations of prf.GLFG52 × 12 and NaCl to determine Tp, which is plotted against prf.GLFG52 × 12 concentration. Data are plotted as mean values ± standard deviations (S.D.s) of three replicates. g Photographs of 30 mg of phase-separated prf.GLFG52 × 12 after centrifugation into a volume of ~150 μl at 25 °C, heated to 65 °C for 1 min, then cooled down on ice for 15 min. The red arrow marks the boundary between the condensed and the aqueous phase before heating.
