title: Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.
abstract: Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76049 Karlsruhe, Germany.
authors.forename: René M
authors.initials: RM
authors.lastname: Dörlich
authors.affiliation: 1] Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany [2] Faculty of Bioscience, University of Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76049 Karlsruhe, Germany.
authors.forename: Per
authors.initials: P
authors.lastname: Niklas Hedde
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76049 Karlsruhe, Germany.
authors.forename: Vittoria
authors.initials: V
authors.lastname: Schuster
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76049 Karlsruhe, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Hippler
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Wesslowski
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany.
authors.forename: Gary
authors.initials: G
authors.lastname: Davidson
authors.affiliation: 1] Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), 76049 Karlsruhe, Germany [2] Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany [3] Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D002460:Cell Line; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal / Q000379:methods; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface