Fig. 4: Epsin1 and ubiquitylation play compensatory roles in endocytic.

a Representative image of a SUM cell expressing gene-edited AP2 σ2-HaloTag: JF646 (cyan) and Eps15-mCherry (red). The scale bars 10 μm. Large inset highlights three representative clathrin-coated structures shown in smaller insets. The scale bars 5 μm. b Representative image of short-lived (blue), intermediate-lived fraction (yellow) and long-lived (green) structures lasting <20 s, > 20 s and > 5 min, respectively. c Histograms of lifetime distributions of clathrin-coated structures under different experimental groups. Lifetime shorter than 20 s is considered short-lived, lifetime between 20 and 180 s is labeled as intermediate-lived fraction and structures lasting longer than 180 s are long-lived. Data are mean ± S.E.M. WT represents SUM cells that have endogenous Eps15 and Epsin1 expression. Eps15 KO represents SUM cells that were CRISPR modified to knock out alleles of endogenous Eps15. Epsin1 KD represents Epsin1 knock down in WT cells using siRNA. Eps15-DUB represents Eps15 knock out cells transfected with Eps15 fused with DUB (to C terminal end of Eps15). Eps15-DUB Epsin1 KD represents Eps15 knock out cells transfected with Eps15 fused with DUB and knocked down for Epsin1. All three constructs have mCherry at their C terminus for visualization. for WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 19, 19, 18 and 15 biologically independent cells were imaged. d Bar plot summarizing percentage of short-lived, intermediate-lived fraction and long-lived fraction for the four conditions. Data are mean ± S.E.M. e Bar plot of the intermediate-lived fraction for all four groups. Data are mean ± S.E.M. f Bar plot of the short-lived fraction for all four groups, zoomed-in representation of the short-lived fraction from panel c. Data are mean ± S.E.M. In panel (d), (e), and (f), for WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 19, 19, 18 and 15 biologically independent cells were imaged. In total > 10000 pits were analyzed for each group. An unpaired, two-tailed student’s t test was used for statistical significance using GraphPad prism. p < 0.05 is considered significantly different. All cell images were collected at 37 °C.