Fig. 1

Flotillins mediate sorting of internalised TCRζ into Rab5 and Rab11a endosomes. a Schematic depicting photoactivation to investigate sorting. Top: sorting of photoactivated endosomes (red) into the Rab compartment (green, yellow = merged), bottom: photoactivated endosomes not sorting into the Rab compartment. Dashed lines: quantified compartment mask. b Representative images of photoactivated (dashed region) TCRζ-PAmCherry sorting into EGFP-Rab4 in WT (left) and FlotKO (right) Jurkat T cells. Cells photoactivated for five frames with 2.5 s intervals, then imaged every 2.5 s for 150 frames. c % endosomal TCRζ-PAmCherry intensity in Rab4 in WT or FlotKO cells. Dashed line represents timepoint where TCRζ-PAmCherry intensity significantly diverges between WT and FlotKO. d Percentage reduction in endosomal TCRζ-PAmCherry intensity in Rab4 from the 50 to 55 s timepoint. Fitted linear regression line is bolded. n.s. indicates slopes are not significantly different. e Representative images of photoactivated (dashed regions) TCRζ-PAmCherry sorting into EGFP-Rab5 in WT (left) and FlotKO (right) cells. Cells imaged as in b. f Percentage endosomal TCRζ-PAmCherry in Rab5 in WT or FlotKO cells over time. g Percentage reduction in endosomal TCRζ-PAmCherry intensity in Rab5 from 50 to 55 s. Fitted linear regression line is bolded. ***Indicates slopes are highly significantly different. h Representative images of photoactivated (dashed region) TCRζ-PAmCherry sorting into EGFP-Rab11a in WT (left) and FlotKO (right) cells. Cells were photoactivated for five frames with 2.5 s intervals, then imaged at 1 frame per minute for 10 min. i % endosomal TCRζ-PAmCherry in Rab11 in WT or FlotKO cells over time. j Percentage of TCRζ-PAmCherry intensity in Rab5 and Rab11 in FlotKO cells compared to WT. *p < 0.05, **p < 0.01 from one sample t-test comparing values to normalised WT mean of 100% j. Dotted line = majority of timepoints after which p < 0.05, dashed line = p < 0.01, solid line = timepoints after which p > 0.05 from Wilcoxon rank sum test. Data points = means of n = 3 (Rab4, WT: 5; 4; 5 cells and FlotKO: 4; 3; 4 cells per experiment), n = 4 (Rab5, WT: 7; 5; 6; 7 cells and FlotKO: 3; 7; 4; 3 cells per experiment) and n = 4 (Rab11, WT: 6; 1; 7; 4 cells and FlotKO: 4; 4; 2; 2 cells per experiment) biologically independent experiments, error bars = mean ± SEM. Scale bar = 5 µm