Fig. 7: Restoring phase separation of Rph3A reinstates its effect on surface clustering and mobility of GluN2A.

a Schematic illustration of dND-fused constructs used to restore phase separation of Rph3A. b OptoDroplet assay of indicated constructs in a. The number of puncta per cell was plotted. Data are displayed as the mean ± SEM (dND: n = 7 cells; dND-IDR1-R9A cells: n = 9 cells; IDR1-R9A: n = 6 cells). c Representative images of EGFP-tagged R9A and dND-fused Rph3A R9A at dendrites of hippocampus neurons. The density and synaptic localization of puncta were quantified. The data are displayed as the mean ± SEM (R9A: n = 6 dendrites from 3 neurons; dND-R9A: n = 9 dendrites from 5 neurons, ^**p < 0.01, two-tailed unpaired t test). d Representative images of the FRAP assay of SEP-GluN2A in Rph3A-knockdown and Rph3A-reexpressing neurons. e Quantification of the fluorescence recovery in d. The data are displayed as the mean ± SEM (Rph3A WT: n = 9 puncta; Rph3A R9A: n = 9 puncta; dND-Rph3A R9A: n = 9 puncta; dND: n = 10 puncta). f The recovery rate of SEP-GluN2A. The data are displayed as the mean ± SEM (n numbers are defined in e, ^*p < 0.05, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). g The mobile fraction of SEP-GluN2A. The data are displayed as the mean ± SEM (n numbers are defined in e, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). h Representative images of surface GluN2A and Homer1 staining in Rph3A-knockdown and Rph3A-reexpressing neurons. i–k Quantification of the percentage of synaptic GluN2A clusters, synaptic and extra-synaptic surface GluN2A cluster fluorescence intensity and density. The data are displayed as the mean ± SEM (n = 10 dendrites from 5 neurons for each group, ^*p < 0.05, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). The full images of c, d and h are showed in Supplementary Fig. 13. Source data and p values of b, c, e, f, g, i, j and k are provided in the Source Data file.