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. Wepresent a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by antiGFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasmamembrane with photostable fluorescence dyes. This labeling technique enables 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 identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule cotracking and quantitative single-molecule Forster resonance energy transfer moreover reveals transient assembly of IFNg receptor heterotetramers and confirms its structural architecture.

Automated summary

Localization and tracking of individual receptors by single-molecule imaging allows for the study of assembly and dynamics of signaling complexes in the plasma membrane. This article presents a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells using up to four colors. The use of engineered GFP variants and fluorescent dyes enables the quantitative resolution of the stoichiometry and dynamics of the IFNg receptor signaling complex in living cells.