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Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy
Antonia Franziska Eckert1, Peng Gao1,2, Janine Wesslowski3
1Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, 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.
Insights
This study presents a new fluorescence microscopy method for precisely measuring Wnt pathway interactions in live cells. The technique accurately quantifies ligand-receptor binding, even with low receptor levels, advancing cell signaling research.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Signaling
Background:
- Multicellular development relies on cell-cell signaling networks.
- Wnt signaling pathways use ligands and receptors for cellular responses.
- Quantitative characterization of these interactions is crucial but challenging.
Purpose of the Study:
- To develop a robust method for quantitatively measuring ligand-receptor interactions in live cells.
- To determine equilibrium dissociation coefficients for Wnt pathway interactions.
- To overcome challenges of live-cell imaging like movement and low signal-to-noise.
Main Methods:
- Utilized fluorescence correlation spectroscopy (FCS) with ultra-high speed axial line scanning.
- Employed CRISPR/Cas9 gene editing to endogenously tag receptors with fluorescent proteins.
- Applied the method to study interactions within the Wnt signaling pathway.
Main Results:
- Achieved precise equilibrium dissociation coefficients for Wnt pathway interactions.
- Demonstrated method's accuracy even with low, near-native receptor concentrations.
- Successfully quantified ligand-receptor binding dynamics in live cells.
Conclusions:
- The developed ultra-high speed FCS method provides precise measurements of ligand-receptor interactions.
- This technique enables quantitative analysis of cell signaling pathways under near-native conditions.
- The approach is valuable for understanding complex biological networks like Wnt signaling.
