Fig. 3
From: Flotillins promote T cell receptor sorting through a fast Rab5–Rab11 endocytic recycling axis
Flotillins regulate TCRζ sorting into Rab11a vesicles for fusion with the plasma membrane. a Representative images of a TCRζ-GFP vesicle fusion event. Zoomed images below represent the indicated fusion event (white box) at 200 ms intervals. Main image scale bar = 5 µm, inset scale bar = 1 µm. b TCRζ-GFP vesicle fusion events per minute in WT and two FlotKO Jurkat T cell lines. c Representative images of TCRζ-GFP (cyan) vesicle fusion events with either flotillin-1 and flotillin-2 mCherry (magenta, top two panels) or mCherry-Rab11a (magenta, bottom panel). Scale bar = 0.5 µm. d Percentage of TCRζ-GFP fusion events containing either flotillin1/2-mCherry or mCherry-Rab11a. e Representative images displaying a GFP-Rab11a vesicle fusion event. Zoomed images below represent the indicated fusion event (white box) at 200 ms intervals. Main image scale bar = 5 µm, inset scale bar = 1 µm. f GFP-Rab11a vesicle fusion events per minute in WT and two FlotKO Jurkat T cell lines. g Representative images of TCRζ-GFP (cyan) vesicle fusion events with mCherry-Rab11a (magenta) in WT Jurkat T cells (top) or fusion with (middle) or without (bottom) mCherry-Rab11a in FlotKO Jurkat T cells. Scale bar = 0.5 µm. h Percentage of TCRζ-GFP fusion events containing mCherry-Rab11a in WT and two FlotKO Jurkat T cell lines. All images captured on a Zeiss ELYRA TIRF microscope. Data points indicate means of independent experiments, error bars indicate mean ± SEM. n.s. = not significant, *p < 0.05, **p < 0.01 from a two-tailed unpaired Student’s T-test of means for TCRζ fusion of n = 6 (WT: 7; 6; 5; 10; 4; 5 cells, FlotKO#1: 3; 6; 3; 4; 6; 4 cells per experiment), 3 (FlotKO#2: 4; 5; 4 cells per experiment), n = 3 (Rab11a fusion, WT: 5; 5; 4 cells, KO#1: 5; 4; 3 cells, KO#2: 5; 5; 5 cells per experiment), 3 (flotillin1/2: 6; 5; 7 cells per experiment), 4 (Rab11a: 5; 5; 4; 5 cells per experiment), and 4 (TCR fusion with Rab11a, WT: 5; 5; 4; 5 cells, KO#1: 5; 5; 3; 5 cells, KO#2: 2; 3; 5; 4 cells per experiment) biologically independent experiments
