Fig. 3: Par6β promoted LLPS of Par3N in vitro.
From: Par complex cluster formation mediated by phase separation
a Protein concentration-dependent LLPS of Par3N or Par3N/Par6β complex. Only Par3N was iFluorTM 488 labeled. The fluorescence imaging settings were identical for easy comparison. Images were acquired at ~2 min after injecting the mixture into the chamber. b Column scatter charts show the droplet size of Par3N (25 µM, n = 250 droplets examined over five independent observation fields) or Par3N/Par6β complex (25 µM, n = 250 droplets examined over five independent observation fields). Data are shown as mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA with Tukey’s multiple comparison test. c, d Representative SDS–PAGE analysis and quantification data showing the distribution of proteins between aqueous solution/supernatant (S) and condensed liquid phase/pellet (P) fractions. Par3N and Par6β were mixed at a 1:0 f or 1:1 g molar ratio at various concentrations. Experiments were performed three times independently with similar results. Data are expressed as mean ± SD. e, f The time-lapse images showing the localization of iFluorTM 488-Par3N e or co-localization of iFluorTM 488-Par3N and Cy3-Par6β complex f in the droplets with enriched concentrations. The enlarged images at right show that small droplets undergo time-dependent coalescence into larger ones. The 0 min images were acquired at ~5 min after injecting the mixture into the chamber. g FRAP analysis of iFluorTM 488-Par3N droplets in the absence or presence of Par6β in vitro showing the exchange kinetics of the protein in droplets with the surrounding aqueous solution. The curves below represent FRAP recovery curves of iFluorTM 488-Par3N (with or without Par6β) by averaging signals of 20 droplets with similar sizes each after photobleaching. Time 0 refers to the time point of the photobleaching pulse. Experiments were performed three times independently with similar results. Data are represented as mean ± SD. Source data are provided as a Source data file.
