Fig 2: Effects of glutamine depletion on HCT116 cells and their EVsPanels show the effect of different treatments on HCT116 colorectal cancer cells and on the EVs they secrete. Bar chart showing relative levels of phosphorylation of mTORC1 downstream readouts, S6 and 4E-BP1 (measured as a ratio of p-S65-4E-BP1 to pan 4E-BP1 or of the ?-phosphorylated form of 4E-BP1 relative to total 4E-BP1), following growth of cells for 24 h in glutamine-depleted (2.00 mM) versus glutamine-replete (0.15 mM) conditions. Data are from triplicate experiments, analysed as in Fig 4A.Nanosight Tracking Analysis of EV size and number for diluted samples (normalised to cell lysate protein levels) produced from ultracentrifugation (UC) of medium from cells cultured in glutamine-replete and glutamine-depleted conditions for 24 h, as in Fig 4B.Electron micrographs (EMs) of EV samples from glutamine-replete and glutamine-depleted cells as analysed in (B). Arrows indicate representative EVs with the characteristic cup-shaped morphology previously reported. Scale bars represent 500 nm (left panels) and 200 nm (right panels).Comparative Western analysis of putative exosome and non-exosome markers in HCT116 cell lysate (cells) and EV preparation produced by UC from cells grown in glutamine-replete conditions (EVs). Lanes are loaded with equal amounts of protein. Note that unlike other “classical” exosome markers, Rab11a and Cav-1 are present, but not enriched in EVs, while Golgi (TGN46), ER (calnexin) and early endosome (EEA1) markers are absent.Bar charts show changes in levels of putative exosome proteins in EVs produced by UC from cells grown in glutamine-depleted versus glutamine-replete conditions and analysed as in Fig 4B. Protein quantities in each sample were normalised to EV particle number, CD81, Syn-1 and Tsg101 in the four graphs (n = 4, except for particle number, where n = 6). All show an increase in Rab11a and Cav-1, and a decrease in CD63, while small or no change is seen for other markers.Western blot analysis of EV preparations isolated from HCT116 cells cultured in glutamine-replete and glutamine-depleted conditions for 24 h using size-exclusion chromatography (SEC; fractions two to four). EV loading was normalised to protein levels in cell lysates. Bar chart shows relative levels of putative exosome markers normalised to cell lysate protein mass (n = 3). (F') Nanosight Tracking Analysis of EV size and number for samples produced as in (F).Data information: Bar charts derived from at least three independent experiments and analysed by the Kruskal–Wallis test; *P < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD. Significantly decreased levels are in blue and increased levels are in red.

Fig 3: Flotillins mediate sorting of internalised TCR? 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) TCR?-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 TCR?-PAmCherry intensity in Rab4 in WT or FlotKO cells. Dashed line represents timepoint where TCR?-PAmCherry intensity significantly diverges between WT and FlotKO. d Percentage reduction in endosomal TCR?-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) TCR?-PAmCherry sorting into EGFP-Rab5 in WT (left) and FlotKO (right) cells. Cells imaged as in b. f Percentage endosomal TCR?-PAmCherry in Rab5 in WT or FlotKO cells over time. g Percentage reduction in endosomal TCR?-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) TCR?-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 TCR?-PAmCherry in Rab11 in WT or FlotKO cells over time. j Percentage of TCR?-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

Fig 4: Flotillins regulate TCR? sorting into Rab11a vesicles for fusion with the plasma membrane. a Representative images of a TCR?-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 TCR?-GFP vesicle fusion events per minute in WT and two FlotKO Jurkat T cell lines. c Representative images of TCR?-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 TCR?-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 TCR?-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 TCR?-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 TCR? 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

Fig 5: TCR? 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 TCR?-PSCFP2 photoactivated with 800 nm light from a two-photon laser, 2.5 µm within the cell at the cell centre-localised TCR?-mCherry compartment displaying recycling to the plasma membrane over time. Dashed circle = photoactivation region. c % increase in photoactivated TCR?-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by TCR?-mCherry in WT or FlotKO Jurkat T cells or cytoplasmic photoactivation. Greyed region = time of photoactivation. d Mean TCR?-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated compartments. e Mean intenisty of photoactivated TCR?-PSCFP2 2 µm within the cell from TCR?-mCherry in WT or FlotKO cells. Greyed region = time of photoactivation. f % increase in photoactivated TCR?-PSCFP2 signal over time at the plasma membrane when activated from cell centre compartments labelled by mCherry-Rab11a, flotillin1/2-mCherry or TCR?-mCherry in WT Jurkat T cells. Grey box = time of photoactivation. Greyed region = time of photoactivation. g Mean TCR?-PSCFP2 signal from 900 to 1200 s timepoints when photoactivated from the indicated intracellular compartments. h Percentage of photoactivated TCR?-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 (TCR?: 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 (TCR? in FlotKO: 1; 1; 2 cells per experiment) biologically independent experiments. Scale bar = 5 µm