Fig. 4
Flotillin expression is required for recycling of TCR but not for its internalization. a Flotillin knock-out Jurkat T cells expressing TCRζ-PA-mCherry, activated on anti-CD3ε-coated and anti-CD28-coated surfaces, photoactivated on outer membrane region of interest, and subsequently imaged for 250 s. b Number of PA-mCherry vesicles detected for each frame during the time of acquisition. c Maximum number of PA-mCherry vesicles detected in a given frame for Flotillin1/2 KO T cells and WT T cells (dotted line). d Diagram of internalization flow cytometry assay. e Mean fluorescent intensity (MFI) at the surface of 50,000 Jurkat T cells (Left: wildtype; Right: Flotillin1/2 KO) stained with an activating antibody against CD3ε and allowed to internalize TCR complexes for 0, 5, 10, and 20 min. f Decrease of MFI at indicated time points relative to t = 0. g Diagram of antibody feeding assay. h MFI of 50,000 Jurkat T cells (Left: wildtype; Right: Flotillin-1/-2 KO) allowed to recycle TCR for 0, 5, 10, and 20 min after having internalizing TCR complexes labelled with an activating antibody against CD30ε for 40 min. i MFI increase at indicated time points relative to t = 0. j Images of WT and flotillin1/2 KO cells on activated surfaces and expressing TCRζ-PA-mCherry at 1 and 10 min after photoactivation. k Number of PA-mCherry vesicles detected at 10 min after photoactivation. Scale bar, 5 µm. Data obtained from three or more independent experiments, with at least five cells per experiment. Small horizontal dotted lines indicate mean (±SEM). ns, not significant; *p < 0.01, Mann–Whitney t-test
