Intracellular overlap CD63 with small-GTPases. (A and B) Fluorescent analysis of Early- (EE), Recycling- (RE), and Late-Endosome (LE)/Lysosome (L)-associated small-GTPases in CD63-pHluorin/pHuji expressing HeLa cells. Representative examples of (A) Rab5a-GFP (EE), Rab11a-mCherry (RE), Rab7a-GFP, Arl8b-mCherry, and Rab27a/b-GFP (LE/L) localization in relation to CD63-pHluorin/pHuji. (B) Quantification of colocalization between CD63 and various small-GTPases as performed in A and B. n ≥ 3 images (3≥ cells per image) analyzed from ≥2 independent experiments. Red line indicates median. (C) Signal pattern for fluorescently tagged RabGTPases averaged over >12 events, synchronized using peak-intensity of the CD63-pHluorin/pHuji signal at 60 s. (D–G) Examples of DC TIRF-M analysis depicted in C for (D) Rab7a-mCherry signal at CD63-pHluorin fusion spot (inset is 8.2 µm), (E) Rab5a-mCherry and Rab11a-mCherry signal at CD63-pHluorin fusion spot (inset sizes are 8.5 and 5.9 µm, respectively), (F) Rab27b-GFP signal at CD63-pHuji fusion spot (inset is 5.1 µm), (G) Arl8b-mCherry and Rab27a-GFP signal at CD63-pHluorin/pHuji fusion spot in HeLa cells (inset sizes are 5.1 µm). Bars: 5 µm.