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

• Published in: Cell reports methods Vol. 2; no. 2; p. 100165
• Main Authors: Sotolongo Bellón, Junel, Birkholz, Oliver, Richter, Christian P., Eull, Florian, Kenneweg, Hella, Wilmes, Stephan, Rothbauer, Ulrich, You, Changjiang, Walter, Mark R., Kurre, Rainer, Piehler, Jacob
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.02.2022; Elsevier
• Subjects: cell surface labeling; cytokine receptor; live-cell imaging; multicolor imaging; plasma membrane dynamics; receptor dimerization; single molecule tracking; single-molecule fluorescence microscopy; single-molecule FRET; type II interferon receptor; type II interferon receptor; receptor dimerization; cytokine receptor; multicolor imaging; live-cell imaging; single-molecule FRET; single-molecule fluorescence microscopy; plasma membrane dynamics; single molecule tracking; cell surface labeling
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2022.100165

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. We present 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 anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) 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 co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture. [Display omitted] •Engineered nanobody targets enable selective and efficient cell surface labeling•Single-molecule co-tracking robustly identifies ligand-induced receptor dimerization•Single-molecule FRET reveals the geometry of signaling complexes in live cells•Simultaneous four-color single-molecule imaging resolves homo- and heterodimerization Unraveling the spatiotemporal organization and dynamics of receptors in the plasma membrane remains a key challenge for our mechanistic understanding of cellular signaling. Many of the current models in the field propose pre-organization at molecular and supramolecular scale. Live-cell single-molecule localization microscopy offers exciting possibilities for scrutinizing such models, but a robust methodology is still lacking. To meet this challenge, we have developed a comprehensive workflow covering high-fidelity labeling as well as dedicated multicolor single-molecule imaging and analysis techniques. Sotolongo Bellón et al. describe a comprehensive workflow for analyzing diffusion and interaction of cell surface receptors by multicolor single-molecule imaging. Based on engineered orthogonal labeling in combination with spatiotemporal cross-correlation techniques and single-molecule FRET, they identify ligand-induced homo- and heterodimerization of the interferon-γ receptor in live cells.