Quantifying Clathrin-Mediated Protein Enrichment

CLSs were detected and quantified using custom software developed in Matlab (MathWorks), as previously described (Aguet et al., 2013; Garay et al., 2015) (64 (link)). Briefly, diffraction-limited clathrin structures were detected using a Gaussian-based model method to approximate the point-spread function of eGFP-CLCa or antibody-labeled clathrin in TIRF-M images. The fluorescence intensity corresponding to the secondary channel such as that of a second label (e.g. CALM, EPSIN, AAK1) or of clathrin within epifluorescence images within CLSs was determined by the amplitude of the Gaussian model for the appropriate fluorescent channel for each structure. As such, the measurements of protein intensity within CLSs represent enrichment of the corresponding signal relative to the local background fluorescence in the vicinity of the detected CLS. Measurements (mean levels of various proteins within specified CLS subset for each cell) were subjected to either two-sided student’s t test or ANOVA followed by Tukey post-test, with a threshold of p < 0.05 for statistically significant differences between conditions.