Unassembled caveolin in the plasma membrane. (A and B) CV1-CAV1-mEGFP cells were left untreated (A) or treated with 5 µg/ml U18666A for 16 h (B), fixed, and viewed by confocal microscopy. U18666A treatment resulted in a noncaveolar pool of CAV1 in the plasma membrane. (right) Insets show enlargements of the boxed areas. (C) FRAP analysis of the noncaveolar surface pool of CAV1. 4 × 4–µm squares were photobleached in the periphery of either CV1-CAV1-mEGFP cells (5 µg/ml U18666A treated for 16 h [n = 10 cells]; and untreated [n = 5 cells]) or a CV1 cell line stably expressing GFP-GPI (n = 15 cells). Error bars indicate mean ± SEM. (D) CV1 cells pretreated with 5 µg/ml U18666A (16 h) were transfected with CAV1-mEGFP and post-Golgi trafficking imaged by TIR-FM time-lapse imaging (1 Hz). The evanescent field was adjusted such that the plasma membrane and part of the Golgi were illuminated. Tubular carriers arriving at the surface and releasing CAV1-mEGFP upon fusion with the plasma membrane are seen. Note that CAV1-mEGFP diffused laterally, and no caveolar spots were left behind (see Video 1). (top) Insets show enlargements of the boxed area. (E–G) Treating CV1-CAV1-mEGFP cells with 5 µg/ml U18666A (16 h) or overexpressing CAV1-mEGFP in CV1 cells (16 h) induced unassembled CAV1 in the plasma membrane and targeting of CAV1-mEGFP to Lamp1-positive LE (single confocal sections). (bottom) Insets show enlargements of the corresponding boxed areas. Arrowheads point to Lamp1-positive LE, indicating absence (E) or presence (F and G) of colocalization with CAV1-mEGFP. Bars: (B, D [bottom], and E–G) 10 µm; (D [top]) 2 µm.