Figure 2.

Epsin Is Required for CCV Scission

(A) Effect of RNAi (siRNA pool1) of epsin proteins on transferrin (Tf) uptake measured by flow cytometry. Clathrin (CHC), AP2, and FCHo proteins depletion were used as positive controls (black bars). The values were normalized to the mean of the control cells (gray bars). The background (cells without Tf) for each cell line is shown (white bars). The number of cells analyzed is displayed on each bar. ^∗∗∗p < 0.0001. Data are the mean ± SD.

(B) Effects of 5 independent pools of siRNA against Epsin1+2+3 (red bars) on Tf uptake and of the rescue of pools 1 and 2 (but not CHC and AP2 RNAi) by coexpression of rat epsin1-RFP (green bars). Experiments were done as in (A). Data are the mean ± SD.

(C) Effect of epsin1+2+3 RNAi on the dynamics of clathrin-coated structures (CCS) and rescue by coexpression of rat epsin1-RFP. CCS labeled by σ2-EGFP. Bar, 5 μm.

(D) Scatter plots of individual lifetimes of CCS from three different cells, measured on data set similar to (C). Median with interquartile range is shown on graph and mean ± SD is written at the bottom, n is the number of events analyzed. ^∗∗∗p < 0.0001.

(E) Fraction of CCS with longer duration than the time series.

(F) Scatter plots of individual maximum fluorescence intensities of CCS from three different cells. Data are presented as in (D), excepted for the Log10 vertical axis. ^∗∗∗p < 0.0001.

(G) Morphological analysis of CCS in BSC1 cells treated or not with epsin1+2+3 RNAi. Representative electron microscopy images for various categories quantified (top). Bars, 100 nm. Coated structures were classified as 1, shallow; 2, invaginated; 3, constricted; and 3^∗, multiheaded. Repartition between the various categories of 70 structures from control (white bars) and 1+2+3 RNAi (black bars) cells is shown. Large image on left and 3^∗ image are from RNAi-treated cells.

(H) Effect of epsin1+2+3 RNAi on recruitment of endogenous dynamin 2 (DNM2^en, green) and clathrin (CLTA^en, red). Bar, 5 μm.

(I) Scatter plots of individual lifetimes (top) and individual maximum fluorescence intensities (bottom) of endogenous dynamin2. Data are presented as in (D) and (F), respectively.

See also Figure S2.