Fig. 4

Flotillins rapidly interact with and maintain the organisation of Rab5 and Rab11a-positive endosomes. a Representative images of flotillin-2-PAmCherry photoactivated at the dashed region failing to interact with EGFP-Rab4 endosomes in WT Jurkat T cells. Cells were photoactivated for five frames with 2.5 s intervals, then imaged every 2.5 s for 60 frames. b Representative images of flotillin-2-PAmCherry photoactivated at the dashed region rapidly interacting with EGFP-Rab5 endosomes in WT Jurkat T cells. Cells were photoactivated and imaged as in a. c Representative images of flotillin-2-PAmCherry photoactivated at the dashed region rapidly interacting with EGFP-Rab11 endosomes in WT Jurkat T cells. Cells were photoactivated and imaged as in a. For a–c zoomed images below are indicated by a white box, main image scale bar = 5 µm, inset scale bar = 1 µm, and flotillin-2-PAmCherry was co-expressed with untagged flotillin-1. All cells imaged on a Zeiss 880 confocal, photoactivation performed at 405 nm. d Quantification of the percentage of photoactivated flotillin-2-PAmCherry present in Rab4, 5 or 11a. e Representative confocal images of endogenous Rab4, Rab5 and Rab11a detected with corresponding antibodies in WT (top) or FlotKO (bottom) Jurkat T cells. Scale bar = 5 µm. f Mean fluorescence dispersion of the indicated markers. Data points indicate mean dispersion from n = 3 biologically independent experiments (Fig. 4a–d, Rab4: 10; 8; 5 cells, Rab5: 10; 12; 10 cells, Rab11a: 9; 11; 10 cells per experiment) (Fig. 4e, f, Rab4 WT: 10; 10; 10 cells, Rab4 KO#1: 9; 11; 10 cells, Rab5 WT: 11; 10; 10 cells, Rab5 KO#1: 10; 10; 10 cells, Rab11a WT: 10; 10; 11 cells, Rab11a KO#1: 10; 10; 10 cells per experiment). g Representative transmission electron microscopy images of DAB-reacted APEX2-GBP co-expressed with TCRζ-GFP in WT (top panel) or FlotKO (bottom panel) Jurkat T cells. Scale bar = 2 µm. h APEX-GBP-labelled vesicle circularity. Data points indicate mean circularity from n = 3 (WT: 4; 3; 1 cells per experiment) and 4 (FlotKO#1: 7; 1; 2; 2 cells per experiment) biologically independent experiments of 1–4 cells. *p < 0.05 from F-test to compare variance. Error bars indicate mean ± SEM. n.s. = not significant. *p < 0.05 from Wilcoxon rank sum test d; n.s. = not significant, *p < 0.05, **p < 0.01 from a two-tailed unpaired Student’s T-test of independent experiment means f. n.s. = not significant from a two-tailed unpaired Student’s T-test of independent experiment means and *p < 0.05 from F-test of variance h