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Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.
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.
Afiliación
  • Sotolongo Bellón J; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Birkholz O; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Richter CP; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Eull F; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Kenneweg H; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Wilmes S; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Rothbauer U; Division of Cell Signalling and Immunology, University of Dundee, School of Life Sciences, Dundee, UK.
  • You C; Pharmaceutical Biotechnology, Eberhard-Karls-University, Tübingen, Germany.
  • Walter MR; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.
  • Kurre R; Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.
  • Piehler J; Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.
Cell Rep Methods ; 2(2): 100165, 2022 02 28.
Article en En | MEDLINE | ID: mdl-35474965
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Imagen Individual de Molécula Idioma: En Revista: Cell Rep Methods Año: 2022 Tipo del documento: Article País de afiliación: Alemania Pais de publicación: Estados Unidos
Texto completo
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Imagen Individual de Molécula Idioma: En Revista: Cell Rep Methods Año: 2022 Tipo del documento: Article País de afiliación: Alemania Pais de publicación: Estados Unidos