Fig. 2
From: Flotillins promote T cell receptor sorting through a fast Rab5–Rab11 endocytic recycling axis
TCRζ recycles from an intracellular compartment labelled by flotillins and Rab11a. a Schematic depicting two-photon photoactivation to measure recycling from intracellular compartments. b Representative confocal images of TCRζ-PSCFP2 photoactivated with 800 nm light from a two-photon laser, 2.5 µm within the cell at the cell centre-localised TCRζ-mCherry compartment displaying recycling to the plasma membrane over time. Dashed circle = photoactivation region. c % increase in photoactivated TCRζ-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by TCRζ-mCherry in WT or FlotKO Jurkat T cells or cytoplasmic photoactivation. Greyed region = time of photoactivation. d Mean TCRζ-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated compartments. e Mean intenisty of photoactivated TCRζ-PSCFP2 2 µm within the cell from TCRζ-mCherry in WT or FlotKO cells. Greyed region = time of photoactivation. f % increase in photoactivated TCRζ-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by mCherry-Rab11a, flotillin1/2-mCherry or TCRζ-mCherry in WT Jurkat T cells. Grey box = time of photoactivation. Greyed region = time of photoactivation. g Mean TCRζ-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated intracellular compartments. h Percentage of photoactivated TCRζ-PSCFP2 vesicles within 320 nm of vesicles of the indicated marker outside of the initial photoactivation region. Data points = means of independent experiments, error bars = mean ± SEM. n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 from a two-tailed unpaired Student’s T-test of means of n = 7 (TCRζ: 3; 3; 1; 1; 1; 1; 1 cells per experiment), 5 (flotillin1/2: 2; 1; 1; 1; 1 cells per experiment), 7 (Rab11a: 1; 1; 2; 1; 1; 1; 1 cells per experiment), 3 (cytoplasm: 3; 1; 2 cells per experiment), and 3 (TCRζ in FlotKO: 1; 1; 2 cells per experiment) biologically independent experiments. Scale bar = 5 µm
