Fig. 3. Mutations in both µHD and F-BAR domains of FCHO1 prevent nucleation of clathrin-coated pits (CCP).

a The dynamics of FCHO1-medicated nucleation of CCP in FCHO1-deficient SK-MEL-2 cells expressing RFP-tagged clathrin light chain from endogenous locus (CLTA^RFP/wt) transduced with either wild-type (upper panel), µHD-mutated (p.R679–middle panel) or F-BAR-mutated (p.A34P–bottom panel) GFP-FCHO1 fusion protein. Two micrometre-wide sections of representative movies are shown. Full movies are available in supplemental materials. In the middle panel, contrast was reduced and brightness was increased as to show a diffused signal of GFP at the plasma membrane. b Time dependence of the fluorescent intensity of FCHO1 (green) and endogenous clathrin (red) averaged from nine independent movies. Only the fluorescence of wild-type but not mutant FCHO1 correlates with clathrin. Each channel was normalised to the background and the initial fluorescence was set to 1. Error bars represent SEM of mean fluorescence intensity, n = 9 biologically independent cells from minimum three independent experiments. c Pearson correlation of FCHO1 and clathrin from individual movies. Pooled data from three independent experiments. Each symbol represents one square region of 25 µm². Up to three regions per cells were quantified. Horizontal lines indicate the median. Statistical analysis of significance was performed using one-way ANOVA test followed by Tukey’s multiple comparison test to assess differences between groups Source data are provided as a Source Data file.