Figure 3.
Constitutive internalization and recycling in stably transfected LLC-PK1 cells expressing BBS-5-HT1AR. A, Cells are incubated 30 min at 37°C with BTX-AF488 and transferrin-A546 (TRF; 10 μg/ml; A1, A2), and in the presence of 0.35 m sucrose (A2) to block clathrin-mediated endocytosis or with 80 μm monensin (A3) to block recycling, respectively. Merge pictures point out the colocalization of BTX (in green) in control condition (A1), whereas both BTX and TRF endocytosis were blocked by high sucrose treatment (A2). B, Internalized BBS-5-HT1AR is measured in control conditions and during inhibition of internalization (high sucrose) or recycling (monensin). Data are presented as mean ± SEM integrated fluorescence corresponding to intracellular BTX-AF488 in four independent experiments (300 cells). C, Colocalization of internalized BTX-labeled-BBS-5-HT1AR with rab5-GFP (C1) and rab4-GFP (C2) after 15 and 30 min of BTX-AF555 incubation at 37°C, respectively. In merged pictures (right), yellow labeling shows BTX and rab-GFP colocalization (arrows). D, Individual internalization or recycling events (arrows) were identified using live confocal microscopy and shown as time lapse. E, Localization of entrapped BTX-AF488 in lysosomes. BBS-5-HT1AR internalized (E1) do not colocalize with lysosome-labeled with LysoTracker (E2) on merged picture (E3). *p < 0.05, **p < 0.001 as compared with control (one-way ANOVA, Dunnett's test). Scale bars: A, 20 μm; C, E, 10 μm; D, 2 μm.