Recruitment of Aux1 and GAK to clathrin-coated vesicles in genome-edited cells. (A) Representative plots of single endocytic events (first three panels) and hotspots (last panel) showing fluorescence intensity traces for CLTA-TagRFP and EGFP-Aux1 (arbitrary units for CLTA; number of molecules for Aux1) imaged at 1-s intervals by TIRF microscopy. (B) Representative plots of single endocytic events (first three panels) and hotspots (last panel) showing fluorescence intensity traces for CLTA-TagRFP and EGFP-GAK imaged at 1-s intervals by TIRF microscopy. (C) EGFP-Aux1 recruitment was not detected while coated pits were assembling. Representative plots of a single endocytic event showing fluorescence intensity traces for CLTA-TagRFP and EGFP-Aux1 (left) imaged at 250-ms intervals by TIRF microscopy. The EGFP-Aux1 signal was detected and measured as indicated in the insert image. Right: Fluorescence intensity fluctuations of the EGFP channel measured from the boxed area 12 pixels away from the detected EGFP-Aux1 burst signal. (D) EGFP-GAK recruitment was not detected while coated pits were assembling. (E) Stepwise recruitment of Aux1 to coated vesicles. Representative plots of EGPF-Aux1 burst-like recruitment (shown as number of molecules for Aux1) imaged at 62.5-ms intervals with TIRF microscopy; fit (red) obtained by applying a step-fitting function to estimate the average recruited molecules during the initiation phase of Aux1 burst-like recruitment. The last two panels show the histogram distributions (with Gaussian fitting) of EGFP-Aux1 molecules during the first step and second step of its recruitment. (F) Stepwise recruitment of GAK to coated vesicles.