Kiuchi Tai, Kobayashi Ryouhei, Ogawa Shuichiro, Elverston Louis L H, Vavylonis Dimitrios, Watanabe Naoki
Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.
Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Sakyo-ku, Kyoto 606-8501, Japan.
Structure. 2025 Jun 5;33(6):997-1006.e6. doi: 10.1016/j.str.2025.03.012. Epub 2025 Apr 23.
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.
超分辨率显微镜可实现几纳米的分辨率，但分子复合物中的共定位分析受其标记密度限制。在此，我们提出一种使用多重超分辨率成像结合可交换单分子定位（IRIS）对分子复合物进行定量映射的方法。我们开发了抗血清衍生的Fab IRIS探针，用于对内源蛋白进行高密度标记和蛋白质簇着色（PC着色），该方法采用基于像素的主成分分析和聚类。PC着色可绘制多种蛋白质不同比例的区域图，并在每个区域计算两种蛋白质之间的相关性以评估复合物的形成。PC着色揭示了内吞作用之前网格蛋白包被结构（CCS）中的多层复合物形成。在表皮生长因子（EGF）刺激后，从CCS边缘外侧起依次排列着以表皮生长因子受体（EGFR）为主的区域、EGFR - Grb2复合物区域和以Grb2为主的区域。沿着以Grb2为主的区域内部，CCS成分（Eps15、FCHo1/2和intersectin - 1）与Grb2形成复合物，远离EGFR。以Grb2为主的区域以及Grb2 - CCS成分复合物的形成可能决定了EGFR在CCS边缘的招募位点。
