Birkholz, Oliver

Eull, Florian

Kenneweg, Hella

Kurre, Rainer

Piehler, Jacob

Richter, Christian P.

Rothbauer, Ulrich

Sotolongo Bellón, Junel

Walter, Mark R.

Wilmes, Stephan

You, Changjiang

English

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.</p

University of Dundee Online Publications

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

https://discovery.dundee.ac.uk/files/72717956/1_s2.0_S2667237522000170_main.pdf

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

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University of Dundee Online Publications

University of Dundee Online Publications