Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. For this purpose, we here developed a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. We engineered two monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies (NB) for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enabled us to quantitatively resolve the stoichiometry and dynamics of the interferon-g (IFNg) receptor signaling complex in the plasma membrane of living cells by multicolor single molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we unambiguously identify ligand-induced receptor homo-and heterodimerization. Detailed analysis by multicolor single-molecule co-tracking and quantitative single-molecule FRET revealed transient assembly of IFNγ-receptor heterotetramers in the plasma membrane with a structural architecture as observed for the isolated ectodomains in vitro.