Fig. 1
T cell activation and signaling promote fast internalization of TCRζ but not of Lck. a Left: Representative images of Jurkat T cells expressing TCRζ-PA-mCherry (top panels) or Lck-PA-mCherry (bottom panels), allowed to adhere on non-activating (poly-l-lysine) or b activating (anti-CD3ε and anti-CD28) surfaces, photoactivated on membrane region of interest and subsequently imaged for 250 s. Images show T cells before and after activation and at indicated time points. Right: number of PA-mCherry vesicles detected in each frame during the time of acquisition. c Maximum number of PA-mCherry vesicles detected in a given frame during the 250 s acquisition time. Each dot represents a cell. d Maximum number of PA-mCherry vesicles detected in Jurkat cells lacking functional Lck (JCam1), Zap70 (P116) or Lat (LAT KO) and activated on anti-CD3ε and anti-CD28 coated surfaces. Horizontal dashed line represents the values for TCRζ in activated cells from panel c. e Representative examples of TCRζ-PA-mCherry vesicle tracks detected in activated untreated cells or treated with dynasore or CK666. Color scale is length in µm. f Length of vesicle tracks in untreated, dynasore or CK666 treated cells. Each dot is one track. g Zoomed images of immobile TCRζ-PA-mCherry vesicles in dynasore (top) or CK666 (bottom) treated Jurkat T cells. Scale bar is 0.5 µm. h Fluorescence intensity profiles over time of single vesicles, such as those shown in g and neighboring plasma membrane regions of the same size. Scale bars, 5 µm. Data obtained from three or more independent experiments. Small horizontal lines indicate mean (±SEM). ns, not significant; *p < 0.01; ***p < 0.0001; ****p < 0.00001; Mann–Whitney t-test
