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

Junel Sotolongo Bellón, Oliver Birkholz, Christian P. Richter, Florian Eull, Hella Kenneweg, Stephan Wilmes, Ulrich Rothbauer, Changjiang You, Mark R. Walter, Rainer Kurre (Lead / Corresponding author), Jacob Piehler (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)
194 Downloads (Pure)


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.

Original languageEnglish
Article number100165
Number of pages11
JournalCell Reports Methods
Issue number2
Early online date4 Feb 2022
Publication statusPublished - 28 Feb 2022


  • 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

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Genetics
  • Computer Science Applications
  • Radiology Nuclear Medicine and imaging


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