Fig. 3
Internalized flotillins and TCRζ define a long-lived mobile endocytic network. a Representative images of Jurkat T cells expressing Clathrin-PA-mCherry (left) or Flotillin-PA-mCherry (right), activated on anti-CD3ε and anti-CD28-coated surfaces, photoactivated on a region of interest and subsequently imaged for 250 s. Images are from 50 and 200 s after photoactivation. Number of PA-mCherry vesicles detected in each frame at (b) 50 s and (c) 200 s. d Number of PA-mCherry vesicles detected at any given point during 250 s acquisition. e Number of PA-mCherry vesicles detected within or outside of the central endosomal region for each frame during the time of acquisition for Flotillin2-PA-mCherry and (f) Clathrin-PA-mCherry. g Ratio of Flotillin2-PA-mCherry or Clathrin-PA-mCherry positive vesicles detected in or out of the central endosomal region. h Example of Jurkat T cells expressing TCRζ-PA-mCherry after photoactivation of a region of interest and imaged for 450 s (extended) or in cells repetitively photoactivated every 8 s (repetitive). i Number of PA-mCherry vesicles detected in each frame during the time of acquisition as in Fig. 1b (standard) or during extended and repetitive acquisition. j Number of TCRζ-PA-mCherry vesicles detected within or outside of the central endosomal region for each frame during the time of acquisition. Each dot represents a cell. Scale bars, 5 µm. Data obtained from three or more independent experiments. Small horizontal lines indicate mean (±SEM). ns, not significant; **p < 0.001, Mann–Whitney t-test
