![Recruitment of Aux1 and GAK to clathrin-coated vesicles in genome-edited cells. (A) CRISPR/Cas9 gene editing strategy used to incorporate EGFP at the N-terminus of Aux1 or GAK. The target sequence at the genomic locus of gene DNAJC6 (Aux1) recognized by the sgRNA, the protospacer adjacent motif (PAM), the start codon ATG (red), and the site of EGFP incorporation upon homologous recombination are highlighted. (B) Schematic representation of the bursts of Aux1/GAK during uncoating of clathrin-coated vesicles (modified from Massol et al., 2006). (C) Snapshot (left) and kymograph (right) from a representative TIRF microscopy time series showing transient burst of EGFP-Aux1 in coated vesicles containing CLTA-TagRFP in SUM159 cell double-edited for EGFP-Aux1+/+ and CLTA-TagRFP+/+. Time series with single molecule detection sensitivity for EGFP acquired for 300 s at 1-s intervals and 100-ms exposures. Kymograph shifted laterally by 5 pixels. Scale bars, 5 µm. (D) Left: Representative plot of a single endocytic event showing fluorescence (F.) intensity traces for CLTA-TagRFP and EGFP-Aux1 (arbitrary units for CLTA; number of molecules for Aux1) together with estimated uncertainties (1 SD, dark shade), corresponding local backgrounds (thin lines), and significance threshold above background (∼2 SD, light shade). Right: Averaged fluorescence intensity traces (mean ± standard error [SE]) for CLTA-TagRFP and EGFP-Aux1 from endocytic coated pits and vesicles automatically identified in eight cells and grouped in cohorts according to lifetimes. Numbers of traces analyzed are shown above each cohort. (E) Distribution of the maximum number of EGFP-Aux1 molecules recruited during the uncoating burst (2,198 traces from 17 cells). (F–H) Transient burst of EGFP-GAK in coated vesicles containing CLTA-TagRFP in SUM159 cell double-edited for EGFP-GAK+/+ and CLTA-TagRFP+/+. Scale bars, 5 µm. Cohorts in G are from eight cells and number of traces analyzed are shown above each cohort; distribution in H is from 1,935 traces from 16 cells. (I) Distribution of the maximum number of EGFP-Aux1 together with EGFP-GAK molecules recruited during uncoating of clathrin-coated vesicles (2,636 traces) from 31 cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (J) Scatter plots for the maximum (max.) fluorescence intensities of EGFP-Aux1 and EGFP-GAK (781 events) as a function of the maximum fluorescence intensity of CLTA-TagRFP (left; Pearson correlation coefficient r = 0.569) or lifetime of clathrin-TagRFP (right; Pearson correlation coefficient r = 0.212) from nine cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (K) Scatter plots of maximum fluorescence intensities of EGFP-Aux1 and EGFP-GAK (716 events) as a function of the duration of Aux1 and GAK bursts (Pearson correlation coefficient r = 0.132) from nine cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (L) Duration of Aux1 and GAK bursts during uncoating of coated vesicles in cells gene-edited for EGFP-Aux1+/+ (six cells, Aux1), EGFP-GAK+/+ (five cells, GAK), or EGFP-Aux1+/+ and EGFP-GAK+/+ (nine cells, Aux1 + GAK) together with CLTA-TagRFP+/+; ***, P < 0.001 by one-way ANOVA with Tukey’s comparison test. (M) Left: Scatter plot of maximum fluorescence intensities and lifetimes from AP2-TagRFP tracks with (996 traces) or without (919 traces) detectable EGFP-Aux1 burst, from nine cells double-edited for EGFP-Aux1+/+ and AP2-TagRFP+/+. Middle: Lifetime distributions from AP2-TagRFP tracks without or with detectable EGFP-Aux1 burst. Right: Fraction of AP2 tracks of lifetime shorter or longer than 20 s with or without detectable EGFP-Aux1 burst. (N) Left: Scatter plot of maximum fluorescence intensities and lifetimes of AP2-TagRFP tracks with (467 traces) or without (350 traces) detectable EGFP-GAK burst, from six cells double-edited for EGFP-GAK+/+ and AP2-TagRFP+/+. Middle: Lifetime distributions from AP2-TagRFP tracks without or with detectable EGFP-GAK burst. Right: Fraction of AP2 tracks of lifetime shorter or longer than 20 s with or without a detectable EGFP-GAK burst.](https://cdn.rupress.org/rup/content_public/journal/jcb/219/3/10.1083_jcb.201908142/3/jcb_201908142_fig1.png?Expires=1767775539&Signature=BgmyN1wS9JLIRwo6DvjTgqkcjRFsV2q56k1KZCu0hY5PSyLtHYWVTV8InRDMhgXjz6xRa-Y0rrlzK2GOnU6cKzBnP4EXyTReCzI43suMfDksr09AyXPgSW~6MV2LshyB~6Vo8Tc--kO9~ZwzITA4Dn7tVGwVoGT9IbuAK6AMRhGDslgWsadIiRjCWUAF2P4hTsTm75PLazVjXHg87-VzlYA~R2c~JCUpGm-CFTECNY7y4ukLgqBCxYFMWI3nvcyhf3UYPkcxd1nyMM~30lEIESlvVIp7EeCvlGG93AM-86E5w4YOTYPklkd5EHKmPVY-urVMu57PGLUoTi6fmMOoAA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Recruitment of Aux1 and GAK to clathrin-coated vesicles in genome-edited cells. (A) CRISPR/Cas9 gene editing strategy used to incorporate EGFP at the N-terminus of Aux1 or GAK. The target sequence at the genomic locus of gene DNAJC6 (Aux1) recognized by the sgRNA, the protospacer adjacent motif (PAM), the start codon ATG (red), and the site of EGFP incorporation upon homologous recombination are highlighted. (B) Schematic representation of the bursts of Aux1/GAK during uncoating of clathrin-coated vesicles (modified from Massol et al., 2006). (C) Snapshot (left) and kymograph (right) from a representative TIRF microscopy time series showing transient burst of EGFP-Aux1 in coated vesicles containing CLTA-TagRFP in SUM159 cell double-edited for EGFP-Aux1+/+ and CLTA-TagRFP+/+. Time series with single molecule detection sensitivity for EGFP acquired for 300 s at 1-s intervals and 100-ms exposures. Kymograph shifted laterally by 5 pixels. Scale bars, 5 µm. (D) Left: Representative plot of a single endocytic event showing fluorescence (F.) intensity traces for CLTA-TagRFP and EGFP-Aux1 (arbitrary units for CLTA; number of molecules for Aux1) together with estimated uncertainties (1 SD, dark shade), corresponding local backgrounds (thin lines), and significance threshold above background (∼2 SD, light shade). Right: Averaged fluorescence intensity traces (mean ± standard error [SE]) for CLTA-TagRFP and EGFP-Aux1 from endocytic coated pits and vesicles automatically identified in eight cells and grouped in cohorts according to lifetimes. Numbers of traces analyzed are shown above each cohort. (E) Distribution of the maximum number of EGFP-Aux1 molecules recruited during the uncoating burst (2,198 traces from 17 cells). (F–H) Transient burst of EGFP-GAK in coated vesicles containing CLTA-TagRFP in SUM159 cell double-edited for EGFP-GAK+/+ and CLTA-TagRFP+/+. Scale bars, 5 µm. Cohorts in G are from eight cells and number of traces analyzed are shown above each cohort; distribution in H is from 1,935 traces from 16 cells. (I) Distribution of the maximum number of EGFP-Aux1 together with EGFP-GAK molecules recruited during uncoating of clathrin-coated vesicles (2,636 traces) from 31 cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (J) Scatter plots for the maximum (max.) fluorescence intensities of EGFP-Aux1 and EGFP-GAK (781 events) as a function of the maximum fluorescence intensity of CLTA-TagRFP (left; Pearson correlation coefficient r = 0.569) or lifetime of clathrin-TagRFP (right; Pearson correlation coefficient r = 0.212) from nine cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (K) Scatter plots of maximum fluorescence intensities of EGFP-Aux1 and EGFP-GAK (716 events) as a function of the duration of Aux1 and GAK bursts (Pearson correlation coefficient r = 0.132) from nine cells triple-edited for EGFP-Aux1+/+, EGFP-GAK+/+, and CLTA-TagRFP+/+. (L) Duration of Aux1 and GAK bursts during uncoating of coated vesicles in cells gene-edited for EGFP-Aux1+/+ (six cells, Aux1), EGFP-GAK+/+ (five cells, GAK), or EGFP-Aux1+/+ and EGFP-GAK+/+ (nine cells, Aux1 + GAK) together with CLTA-TagRFP+/+; ***, P < 0.001 by one-way ANOVA with Tukey’s comparison test. (M) Left: Scatter plot of maximum fluorescence intensities and lifetimes from AP2-TagRFP tracks with (996 traces) or without (919 traces) detectable EGFP-Aux1 burst, from nine cells double-edited for EGFP-Aux1+/+ and AP2-TagRFP+/+. Middle: Lifetime distributions from AP2-TagRFP tracks without or with detectable EGFP-Aux1 burst. Right: Fraction of AP2 tracks of lifetime shorter or longer than 20 s with or without detectable EGFP-Aux1 burst. (N) Left: Scatter plot of maximum fluorescence intensities and lifetimes of AP2-TagRFP tracks with (467 traces) or without (350 traces) detectable EGFP-GAK burst, from six cells double-edited for EGFP-GAK+/+ and AP2-TagRFP+/+. Middle: Lifetime distributions from AP2-TagRFP tracks without or with detectable EGFP-GAK burst. Right: Fraction of AP2 tracks of lifetime shorter or longer than 20 s with or without a detectable EGFP-GAK burst.