Confocal microscopy. (A–D) Immunofluorescence staining for α-adaptin (A and B) and clathrin light chain (C and D) indicates an increase of clathrin-coated pits (CCPs) number in cells that lack all three epsins. In TKO cells, clathrin-coated pits generally occur in small clusters. Insets show the boxed regions at high magnification. Note the large size of TKO cells relative to control. (E) CCP number in WT and TKO cells as assessed by α-adaptin immunofluorescence (***p < 0.001, Student's t test, n = 10 cells/genotype). (F) Kymographs from a time series of WT and TKO cell expressing μ2-adaptin-GFP. Each line represents a single μ2-GFP spot. Note the short length of the lines for WT, reflecting the turnover of the pits and the continuous lines in TKO cells, reflecting an arrest of the pit maturation. (G) Clathrin-coated pit turnover (appearance and disappearance events) as analyzed by spinning-disk confocal imaging of μ2-adaptin-GFP fluorescence in WT and TKO cells (n = 5 cells/genotype). (H and I) Impaired uptake of pre-bound Alexa594-transferrin (Tf) in TKO cells during a 15-min incubation. In WT, the bulk of Tf was internalized, while in TKO Tf remained at the cell surface. (J and K) Transferrin receptor (TfR)-GFP predominantly localizes in intracellular vesicles in WT but at the cell surface in TKO cells. (L) A surface biotinylation assay reveals elevated amounts of endogenously expressed TfR at the plasma membrane of TKO cells relative to WT, as assessed by anti-TfR immunoblotting of streptavidin affinity-purified material. (M–P) Increased surface localization of stably expressed Syb2-HA in TKO fibroblasts as shown by total (M–O) and surface-only (N–P) immunofluorescence. (Q and R) A surface biotinylation performed as in (L) demonstrating an increased fraction of cell surface exposed Syb2-HA in TKO cells relative to WT (**p < 0.01, Student's t test, n = 4 experiments, Surf: surface, Int: internal). (S and T) Representative electron microscopy images of different stage endocytic clathrin-coated intermediates in TKO cells (S) and quantification of the corresponding stages (T, **p < 0.01, ***p < 0.001, n = 33 cells/genotype, one-way ANOVA). (U) Comparative analysis of the localization of clathrin immunoreactivity (CLC) with the localization of dynamin 2, endophillin 2, and myosin 1E immunoreactivities. In WT cells, these three proteins co-localize with a subset of clathrin-coated pits (examples are indicated by small white arrows), which represent late-stage pits. In TKO cells, where more numerous clathrin-coated pits are observed, the punctate localization of dynamin 2, endophillin 2, and myosin 1E is completely lost. Scale bars: 10 μm for (A–D, H–K), 20 μm for (M–P), 5 μm for (U), and 200 nm for (S). In E, G, R, and T black bars indicate WT and red bars epsin TKO. See also Figure 2—figure supplement 1.