2025-04-24 京都大学
<関連情報>
- https://www.kyoto-u.ac.jp/ja/research-news/2025-04-24
- https://www.kyoto-u.ac.jp/sites/default/files/2025-04/web_2504_Kiuchi-a23ce03a19f6d0e50a65ff59e5bae6e0.pdf
- https://www.cell.com/structure/abstract/S0969-2126(25)00132-7
ナノスケールでのタンパク質の共局在化により明らかになったクラスリンコートの分子複合体の層状組織化 Laminar organization of molecular complexes in a clathrin coat revealed by nanoscale protein colocalization
Tai Kiuch ∙ Ryouhei Kobayashi ∙ Shuichiro Ogawa ∙ Louis L.H. Elverston ∙ Dimitrios Vavylonis ∙ Naoki Watanabe
Structure Published:April 23, 2025
DOI:https://doi.org/10.1016/j.str.2025.03.012
Graphical abstract
Highlights
•IRIS probes from antiserum can label antigens beyond the maximum density of antibody
•High-density labeling allows quantitative localization of eight endogenous proteins
•PC-coloring colors regions of distinct ratios of proteins to map their complexes
•PC-coloring and correlation analysis reveal multi-layered complex formation
Summary
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.
