Fig. 5: Loss of Epsin1 and ubiquitylation from endocytic sites rescues transferrin uptake.

a Illustration showing TfR uptake at the plasma membrane. b Flow cytometry histogram of the transferrin fluorescence intensity (green fluorescence) of cells in each condition. n = 3 biological replicates. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. P < 0.05 is considered significantly different. WT represents SUM cells that have endogenous Eps15 and Epsin1 expressions. 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 wild type Eps15 fused with DUB (deubiquitylase fused to C terminal end of Eps15). Eps15-DUB Epsin1 KD represents Eps15 knock out cells transfected with wild type Eps15 fused with DUB and knocked down for Epsin1. All three constructs have mCherry at their C terminus for visualization. c Bar graph represents transferrin fluorescence intensity measured by flow cytometry. Data are mean ± standard deviation, calculated from n = 3 biological replicates. A.U. represents arbitrary units. Flow cytometry runs were carried out at 24 °C. d Histograms of the AP2 intensity distribution tracked in endocytic pits under different experimental groups. The black line indicates the median of the wild-type (WT) cells. For WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 20, 17, 16 and 16 biologically independent cells were used. Total of 6745 and 6034 pits were analyzed for WT, Epsin1 KD. For Eps-DUB and Eps-DUB Epsin1 KD, > 10000 pits were analyzed. e Plots the fraction of endocytic structures that have an AP2 intensity greater or equal to that of a median intensity structure in wild-type (WT) cells. Data are mean ± S.E.M. For WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 20, 17, 16 and 16 biologically independent cells were used. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. P < 0.05 is considered significantly different. f Shows the density (number per area) of endocytic tracks (lifetime between 20 − 180 sec) over 10 minutes for different experimental groups. For WT, Epsin1 KD, Eps-DUB and Eps-DUB Epsin1 KD, n = 19, 19, 16 and 16 biologically independent cells were imaged. An unpaired, two tailed student’s t test was used for statistical significance using GraphPad Prism. P < 0.05 is considered significantly different. g–h Schematic showing how polyubiquitin stabilizes the endocytic protein network by interacting with Eps15 and Epsin1 and how Epsin1 functions as a checkpoint for ubiquitylated cargo. g In presence of polyubiquitin Eps15 and Epsin1 forms a liquid-like initiator network resulting in more stable endocytic structures thereby more productive clathrin-mediated endocytosis. h In absence of ubiquitylated cargo Epsin1 interacts with Eps15 and prevents the assembly of the liquid-like network of Eps15 and thereby acting as a checkpoint for ubiquitylated cargo to prevent unproductive endocytic events.