Laminar organization of molecular complexes in a clathrin coat revealed by nanoscale protein colocalization

Super-resolution microscopy achieves a few nanometers resolution, but colocalization analysis in a molecular complex is limited by its labeling density. Here we present a method for quantitative mapping of molecular complexes using multiplexed super-resolution imaging, integrating exchangeable single-molecule localization (IRIS). We developed antiserum-derived Fab IRIS probes for high-density labeling of endogenous proteins and protein cluster coloring (PC-coloring), which employs pixel-based principal component analysis and clustering. PC-coloring maps regions of distinct ratios of multiple proteins, and in each region, correlation between two proteins is calculated for evaluating the complex formation. PC-coloring revealed multi-layered complex formation in a clathrin-coated structure (CCS) prior to endocytosis. Upon epidermal growth factor (EGF) stimulation, EGF receptor (EGFR)-dominant, EGFR-Grb2-complex, and Grb2-dominant regions lined up from outside the CCS rim. Along the interior of Grb2-dominant regions, CCS components (Eps15, FCHo1/2 and intersectin-1) formed a complex with Grb2 away from EGFR. The Grb2-dominant region and Grb2-CCS component complex formation probably determine EGFR recruitment sites in the CCS rim.