Fig. 4: Delayed immobile cluster formation of βarr in the NC-site-mutant βarr.
From: Membrane-domain compartmentalization of active GPCRs by β-arrestins through PtdIns(4,5)P2 binding
a, Representative TIRF microscopic image of Δβarr1/2 cells expressing EGFP–βarr2 (WT, C-3Q or NC-4Q), SF650-labeled V2R (cyan) and JF549-labeled PLCδ-PH (magenta) before AVP stimulation (basal), after 5-min AVP stimulation and after 15-min AVP stimulation. Scale bar, 3 µm. b, Enlarged view of a and representative trajectories of four diffusion states (immobile, cyan; slow, yellow; medium, green; fast, magenta) of βarr2, V2R and PLCδ-PH molecules after 15-min AVP stimulation. Scale bars, 0.5 µm (βarr2) and 1 µm (V2R and PLCδ-PH). c–e, Heat map showing the time course of intensity histograms for each diffusion state in βarr2 (c), V2R (d) and PLCδ-PH (e) molecules. The particle density with each intensity was displayed according to a color scale. f–h, The difference in fractions of the four diffusion states in βarr2 (f), V2R (g) and PLCδ-PH (h) molecules between basal conditions and after 15-min ligand stimulation. WT, C-3Q and NC-4Q are colored in black, blue and red, respectively. WT AVP (−), n = 19; WT AVP (+), n = 36; C-3Q AVP (−), n = 19; C-3Q AVP (+), n = 32; NC-4Q AVP (−), n = 18; NC-4Q AVP (+), n = 23; n represents individual cells analyzed across two independent biological replicates. AVP (−), vehicle stimulation; AVP (+), AVP stimulation. In a,b, representative images from the groups are shown. In c–e, bars and error bars represent the mean and s.e.m., respectively. Statistical significance was calculated using a two-way ANOVA followed by Tukey’s test for multiple-comparison analysis. *P < 0.05, **P < 0.01 and ***P < 0.001.
