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.
Highlights
Cellular communication is crucial for the development and homeostasis of multicellular organisms
From the time sequence of the intensities (Fig. 2a), all 16 pair correlation functions are calculated (Fig. 2b,c), from which the diffusion coefficients and molecule concentrations are extracted in a global fit (Supplementary Information, Text S5)
In our 2c2f lsFCS measurements, we have found different binding affinities of Dkk[1] and Dkk[2] toward low-densitylipoprotein-receptor related protein 6 (LRP6), which correlate well with the differences observed in their ability to inhibit LRP6 phosphorylation and Wnt/β-catenin signaling (Fig. 3), with the higher-affinity Dkk[1] having a stronger antagonistic effect on LRP6
Summary
Cellular communication is crucial for the development and homeostasis of multicellular organisms. It is clearly important to study ligand-receptor interactions directly in living cells, tissues and entire organisms To this end, fluorescence correlation spectroscopy (FCS) has emerged as a powerful www.nature.com/scientificreports/. A cross-correlation signal is observed if the two molecules bind to each other so that they diffuse as an entity These two methods have been successfully employed on living cells[8] and organisms[9], they are extremely challenging because of the intrinsic movements within biological samples. As in dual-focus FCS, a reference measurement can be avoided by scanning the focus along two parallel lines with known separation and cross-correlating the intensities from the two intersections with the membrane (Supplementary Information, Text S3 and Fig. S3). As an added benefit, alternating excitation with two colors eliminates spectral cross-talk effects
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