CAV1 is exposed to the acidic lumen of LE/LYS. (A) CAV1-mEGFP and CAV1-mCherry colocalized in caveolar spots in the plasma membrane (dashed inset, contrast adjusted) but not in many of the intracellular organelles when imaged live. (B) CV1 cells expressing CAV1-mCherry were stained with 100 nM LysoTracker (green) for 1 h and imaged live to identify acidic organelles. Most acidic organelles were positive for CAV1-mCherry. (C) Colocalization between CAV1-mEGFP and CAV1-mCherry in endosomes was restored by treatment of cells with 0.2 µM BafA (12 h), indicating acid quenching of mEGFP fluorescence. (A–C) Single confocal sections of live cells. (top) Insets show enlargements of boxed areas. (D and E) Live cell time-lapse imaging of CV1 cells coexpressing CAV1-mEGFP and CAV1-mCherry to monitor acid-dependent quenching of CAV1-mEGFP fluorescence during endosomal maturation. (D) An endosome initially positive for CAV1-mEGFP and CAV1-mCherry was tracked over 42 min as outlined in E, and fluorescence intensity profiles were plotted against time. CAV1-mEGFP fluorescence decayed over time, whereas CAV1-mCherry fluorescence was stable. To rule out photobleaching of mEGFP, the total perinuclear mEGFP fluorescence was measured and plotted (E, blue dashed outline; D, intensity profile). Time-lapse series were recorded with epifluorescence illumination at 0.2 Hz. (F) Stills of the CAV1-mEGFP and CAV1-mCherry–positive endosome tracked in D (Video 3). Bars: (A–C) 10 µm; (E) 5 µm; (F) 0.5 µm.