Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy
- Karlsruhe Institute of Technology, Germany
Abstract
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.
Data availability
All data reported in the paper are included in the manuscript and/or supplementary Materials. Source data files have been provided for Figures 2, 5, 6 and 7.
Article and author information
Author details
Gerd Ulrich Nienhaus
Funding
Deutsche Forschungsgemeinschaft (SFB 1324,project A6,project Z2)
- Gerd Ulrich Nienhaus
Deutsche Forschungsgemeinschaft (SFB 1324,project A6)
- Gary Davidson
Helmholtz-Gemeinschaft (STN)
- Gerd Ulrich Nienhaus
Helmholtz-Gemeinschaft (BIFTM)
- Gary Davidson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Eckert et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy
eLife 9:e55286.
https://doi.org/10.7554/eLife.55286
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