Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Nanotechnology, Karlsruhe Institute of Technology, Karlsruhe, Germany
Janine Wesslowski
Institute of Biological and Chemical Systems, Karlsruhe Institute of Technology, Karlsruhe, Germany
Xianxian Wang
Institute of Biological and Chemical Systems, Karlsruhe Institute of Technology, Karlsruhe, Germany
Jasmijn Rath
Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany
Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Nanotechnology, Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Biological and Chemical Systems, Karlsruhe Institute of Technology, Karlsruhe, Germany; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States
Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.
