Extended Data Fig. 5: Controls for Eps15-Cry2 cell experiments.

a, Left: whole cell lysates from WT SUM159/AP-2-HaloTag cells and WT SUM159/AP-2-HaloTag cells gene edited by CRISPR to disrupt Eps15 were separated by SDS-PAGE and immunoblotted for Eps15 and GAPDH. Right: whole cell lysates from WT SUM159/AP-2-HaloTag cells and WT SUM159/AP-2-HaloTag cells transfected with siRNA against Eps15R were collected 24 hours post-transfection. Proteins were separated by SDS-PAGE and immunoblotted for Eps15R and GAPDH. b, Itsn1-mCherry (upper panels), Fcho1-mCherry (lower panels), Eps15-Cry2-GFP, and AP2-HaloTag conjugated to JF₆₄₆ colocalize in Eps15∆ cells expressing Eps15-Cry2 and exposed to low blue light levels. White arrowheads indicate examples of colocalization in endocytic structures. Notably, Fcho1-mCherry/Eps15-Cry2-GFP co-expression often resulted in the formation of large, persistent aggregates on the plasma membrane, denoted by yellow arrowheads. Scale bar is 5 µm. c, The lifetime distributions of AP-2 σ2-HaloTag-labeled endocytic structures in Eps15 knockout cells expressing Eps15-mCherry and in wild-type Eps15 cells are nearly identical. d, The average plasma membrane fluorescence intensity of AP-2 σ2-HaloTag::JF₆₄₆ and Eps15-mCherry in the first frame of each movie analysed in a and Fig. 4. Eps15∆ n=10 biologically independent cell samples, 25,269 pits. WT Eps15 n=10 biologically independent cell samples, 8,969 pits. No light n=11 biologically independent cell samples, 21,996 pits. Low light n=17 biologically independent cell samples, 14,222 pits. Strong light n=12 biologically independent cell samples, 13,978 pits. e, Lifetime distributions of clathrin-coated structures in Eps15∆/Eps15R knockdown cells expressing Eps15-Cry2 at no, low, or strong blue light exposure. Plots show frequency of short-lived (<20 s, magenta), productive (20-180 s, gray), and long-lived (>180 s, yellow) structures for each condition. No blue light exposure resulted in 42 ± 3% (SEM) of CCPs being short-lived (<20 s). Low blue light exposure significantly reduced the frequency of short-lived CCPs from 42 ± 3% to 36 ± 4% (t-test, p=0.042, n=5, 5). While 2-3% of pits were long-lived (>180 s) in cells exposed to no or low blue light, the frequency of long-lived pits increased significantly to 6 ± 1% (t-test, p=0.009, n=5, 5) in cells exposed to strong blue light. No light n=5 biologically independent cell samples, 31,427 pits. Low light n=5 biologically independent cell samples, 27,026 pits. Strong light n=5 biologically independent cell samples, 17,623 pits. f, The lifetime distributions of AP-2 σ2-HaloTag-labeled endocytic structures in Eps15 knockout cells expressing Eps15-mCherry exposed to either no blue light or 50 µW “strong” blue light are nearly identical. g Plot from Fig. 6f and (h) plot from Fig. 6h displaying the individual data points that were averaged together for each FRAP curve. n=5-6 biologically independent samples. Data are presented as mean ± SEM. See Source Data Extended Data Fig. 5.

Source data