Nanoscale cluster mapping of molecular complexes with pixel-based correlations

Super-resolution microscopy achieves a few nanometers resolution, but protein quantification and colocalization is limited by its labeling density. Here we present a method to map molecular complexes using the multiplexed data of IRIS super- resolution imaging. We developed antiserum-derived Fab IRIS probes for high- density labeling of endogenous targets, and image analysis, Protein Cluster coloring (PC-coloring), which employs pixel-based principal component analysis and clustering. PC-coloring was first used to map regions of distinct ratios of targets on the image. The molecular complex formation was then evaluated by correlation between all combinations of two proteins in each PC-colored region. We elucidate receptor recruitment and the complex formation in a clathrin-coated structure (CCS) which is pre-assembled prior to endocytosis. Upon EGF stimulation, EGFR-, EGFR-Grb2-complex- and Grb2-dominant regions lined up from the CCS rim. Along the interior of Grb2-dominant regions, Eps15, FCHo1/2 and intersectin-1 formed a complex with Grb2. The interior of Eps15-FCHo1/2 complexes was lined with Eps15-intersectin-1 complexes. The results reveal a laminar complex formation of EGFR, Grb2 and CCS components at the recruitment sites.