Fig. 2
TCRζ is internalized through a clathrin-independent pathway in vesicles positive for flotillin-2. a Representative images of activated Jurkat T cells expressing TCRζ-PA-mCherry either pretreated with 6 µM pitstop2 or co-expressing AP180-C-EGFP and activated and photoactivated as before. b Maximum number of PA-mCherry vesicles in cells treated with pitstop2 or co-expressing AP180-C-EGFP. c–f Representative images (c and d) and cross-channel nearest neighbor distance (e and f) between vesicles defined by Clathrin-EGFP and TCRζ-PA-mCherry or Clathrin-PA-mCherry and transferrin-Alexa488, and flotillin-2-EGFP and TCRζ-PA-mCherry or flotillin-2-PA-mCherry and transferrin-Alexa488, in Jurkat T cells activated on anti-CD3ε and anti-CD28-coated surfaces. Percentages shown are for vesicles having a distance of less than 320 nm to their nearest neighbor in the other channel. g Representative examples of Clathrin-PA-mCherry (left) or flotillin-2-PA-mCherry (right) vesicle tracks detected in activated T cells untreated (top) or treated with CK666 (bottom). Color scale depicts length in µm. h Length of vesicle tracks in vehicle-treated and CK666-treated cells. Each dot represents a single track. Scale bars, 5 µm. Data obtained from three or more independent experiments. Small horizontal lines indicate mean (±SEM). ns, not significant; **p < 0.001; ****p < 0.00001; Mann–Whitney t-test
