Fig. 6: Disruption of the phase separation capacity of Rph3A influenced the mobility, surface clustering, and synaptic localization of GluN2A in hippocampal neurons.

a Representative images of EGFP-tagged WT and R9A Rph3A at dendrites of hippocampal neurons. Yellow triangles indicate synaptic puncta, and white triangles indicate extrasynaptic puncta of Rph3A. The density, size, and synaptic localization of the puncta of WT and R9A Rph3A, and the sizes of synaptic and extrasynaptic WT Rph3A puncta were also quantified. The data are displayed as the mean ± SEM (WT: n = 10 dendrites from 5 neurons; R9A: n = 6 dendrites from 3 neurons, ^**p < 0.01, two-tailed unpaired t test). b Representative images and quantification of fluorescence recovery from the FRAP analysis of Rph3A puncta at the dendrites of hippocampal neurons. The data are displayed as the mean ± SEM (n = 11 puncta). c Representative images from the FRAP assay of SEP-GluN2A in Rph3A-knockdown and Rph3A-reexpressing (WT and R9A) hippocampal neurons. d Quantification of the fluorescence recovery shown in c. The data are displayed as the mean ± SEM (Scramble: n = 17 puncta; Rph3A shRNA: 20 puncta; Rph3A shRNA-Rph3A WT: 18 puncta; Rph3A shRNA-Rph3A R9A: 20 puncta). e The recovery rate was calculated as the half-time of the recovery curve. The data are displayed as the mean ± SEM (n numbers are defined in d, ^*p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test). f The mobile fraction of GluN2A was calculated based on the plateau of the recovery curve. The data are displayed as the mean ± SEM (n numbers are defined in d, ^**p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test). g Representative images of surface GluN2A and Homer1 staining in Rph3A-knockdown and Rph3A-reexpressing neurons. h-j Quantification of synaptic and extrasynaptic surface GluN2A cluster fluorescence intensity and density and the percentage of synaptic GluN2A clusters. 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 Dunnett’s multiple comparisons test (h, i) or by Tukey’s multiple comparisons test (j)). k Representative trace of NMDA eEPSCs in Rph3A-knockdown and Rph3A-reexpressing neurons. l Quantification of NMDA eEPSCs in Rph3A-knockdown and Rph3A-reexpressing neurons. The data are displayed as the mean ± SEM (Scramble: n = 58 neurons; Rph3A shRNA: 25 neurons; Rph3A shRNA-Rph3A WT: 22 neurons; Rph3A shRNA-Rph3A R9A: 19 neurons, ^*p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test). The full images of a, b, c, g are showed in Supplementary Fig. 13. Source data and p values of a, b, d, e, f, h, i, j and l are provided in the Source Data file.