(A–C) Global lifetime distribution of AP-2 subunit µ2-GFP spots (reflecting endocytic clathrin-coated pits), as assessed by TIRF microscopy in control (A), patient cells (B) and patient cells rescued with mCherry-OCRLWT (C) (Ordinate represents the relative frequency spots at each time point). Histograms were fitted with a Gaussian mixture model identifying three subpopulations of endocytic clathrin-coated pits: short- (mean 40 s, blue), medium- (mean 108 s, green) and long-lived (mean 244 s, red). Note the increase in size of the long-lived subpopulation (red) in patient cells (B), which is rescued by the re-expression of mCherry-OCRLWT (C), (n of events = 8657, 9605, and 6930 respectively; n of cells examined = 12, 12 or 7, respectively). (D–F) Spinning disk analysis of endocytic clathrin-coated pits in control and patient fibroblasts expressing RFP-CLC and GFP-OCRLWT or GFP-catalytically dead OCRL (GFP-OCRLD523G). Representative images are shown in the left panels, kymographs in the middle panels and tracings of the average time courses of fluorescence in the right panels (mean ± SEM, n = 45 events per condition, 3 separate experiments). In control cells (D), there is little overlap between red (clathrin) and predominantly green (OCRL) spots, indicating OCRL recruitment when clathrin fluorescence has started to dim. The signal for OCRL then rapidly disappears as the free vesicle moves away from the imaging plane. The same pattern can be observed in patients cells rescued with OCRLWT (F), while in patient cells expressing OCRLD523G, clathrin lingers for longer times even after the recruitment of OCRL, leading to a substantial overlap of red and green spots (E). Scale bar full size: 2 µm; kymograph: 1 min (red: clathrin; green: OCRL). (G) Colocalization of clathrin and OCRL fluorescence on individual spots from images such as those shown in the left fields of (D–F) as determined by a colocalization plugin on ImageJ (‘Materials and methods’) and reported as Pearson's correlation coefficient (mean ± SEM, n = 7 different cells, 4 separate experiments; two tailed student's t test, ** denotes p = 0.0014 and *** denotes p = 0.0001). (H) Representative images (left) and quantification (right) of different stages of clathrin-mediated endocytosis showing a striking increase in the number of U-shaped pits and coated vesicles per μm of cell perimeter (n = 60 cells; two tailed student's t test, ** denotes p = 0.001, *** denotes p = 0.0001 and **** denotes p = 0.00001). (I and J) A mid-plane confocal image of control (I) and patient (J) cells showing the internalization of Alexa594-labeled transferrin (Tf) that occurs in 2 min. Note that in patient cells transferrin remains mostly at the cell surface. Scale bar: 10 µm. (K) Time course of biotinylated-transferrin internalization in control and patient cells measured by an ELISA-based assay. Uptake is represented as percent of total surface bound biotin-transferrin at 4°C. (n = 3 experiments; two tailed student's t test, ** denotes p = 0.001, and **** denotes p = 0.00001). (L) Internal and surface exposed Tf receptor in control and patient cells as revealed by a biotinylation assay, demonstrating the increase of the surface pool of receptor in patient cells.