Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

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

Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants

Development of novel antimalarials targeting the plasmodial lactate transporter (PfFNT) through a fluorescence cross-correlation spectroscopy-based approach and functional assay

Drug resistance is a significant obstacle in the fight against malaria, prompting the exploration of new treatment approaches and drug targets. One potential target is the recently discovered plasmodial lactate transporter, PfFNT (short for Plasmodium falciparum formate-nitrite transporter). Several studies have shown that inhibiting this transporter can cause the accumulation of toxic levels of lactate within the parasite, leading to its death. Two compounds, BH296 and BH267.meta, have been developed to target both the PfFNT wild type (wt) and a relevant mutant, G107S. So far, these compounds have only been tested using a yeast-based functional assay, and biophysical characterization was missing. In this study, fluorescence cross-correlation spectroscopy (FCCS) measurements were performed to determine true Ki-values, as well as kon and koff rate constants for the binding of inhibitors to both the PfFNT wt and G107S mutant. Once the reliability of FCCS measurements had been confirmed, a compound library of 2,000 inhibitors was screened to identify novel scaffolds that have the potential to inhibit PfFNT. FCCS only allows the search for new inhibitors that displace the existing inhibitor, i.e., share the same binding pocket. To identify an alternative binding site, a functional screening assay with AZD3965 and Metformin was developed in this thesis