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

Eckert, Antonia Franziska; Gao, Peng; Wesslowski, Janine; Wang, Xianxian; Rath, Jasmijn; Nienhaus, Karin; Davidson, Gary; Nienhaus, G. Ulrich

doi:10.7554/elife.55286

Cited by 28 publications

(37 citation statements)

References 45 publications

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“…Created with BioRender.com. more reliable quantitative measurements of molecular dynamics and biochemical reactions in living cells, as recently proposed (Eckert et al, 2020).…”

Section: Discussionmentioning

confidence: 98%

Fast Diffusion Sustains Plasma Membrane Accumulation of Phosphatase of Regenerating Liver-1

Castro-Sánchez

Hernández-Pérez

Aguilar-Sopeña

et al. 2020

Front. Cell Dev. Biol.

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It has been proposed that the accumulation of farnesylated phosphatase of regenerating liver-1 (PRL-1) at the plasma membrane is mediated by static electrostatic interactions of a polybasic region with acidic membrane lipids and assisted by oligomerization. Nonetheless, localization at early and recycling endosomes suggests that the recycling compartment might also contribute to its plasma membrane accumulation. Here, we investigated in live cells the dynamics of PRL-1 fused to the green fluorescent protein (GFP-PRL-1). Blocking the secretory pathway and photobleaching techniques suggested that plasma membrane accumulation of PRL-1 was not sustained by recycling endosomes but by a dynamic exchange of diffusible protein pools. Consistent with this idea, fluorescence correlation spectroscopy in cells overexpressing wild type or monomeric mutants of GFP-PRL-1 measured cytosolic and membrane-diffusing pools of protein that were not dependent on oligomerization. Endogenous expression of GFP-PRL-1 by CRISPR/Cas9 genome edition confirmed the existence of fast diffusing cytosolic and membrane pools of protein. We propose that plasma membrane PRL-1 replenishment is independent of the recycling compartment and the oligomerization state and mainly driven by fast diffusion of the cytosolic pool.

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“…Created with BioRender.com. more reliable quantitative measurements of molecular dynamics and biochemical reactions in living cells, as recently proposed (Eckert et al, 2020).…”

Section: Discussionmentioning

confidence: 98%

Fast Diffusion Sustains Plasma Membrane Accumulation of Phosphatase of Regenerating Liver-1

Castro-Sánchez

Hernández-Pérez

Aguilar-Sopeña

et al. 2020

Front. Cell Dev. Biol.

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“…Conventional two-color scanning FCCS has been previously applied to quantify receptor-ligand interactions in living zebrafish embryos (12) and CRISPR/Cas9 edited cell lines to study such interactions at endogeneous protein level (45). SFSCS is thus directly applicable in the complex environment of living multicellular organisms.…”

Section: Discussionmentioning

confidence: 99%

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

Dunsing

Petrich

Chiantia

2020

Preprint

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Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify transport dynamics and interactions between biomolecules in living cells. For example, cross-correlation analysis of spectrally separated fluctuations allows the investigation of inter-molecular interactions. This analysis is conventionally limited to two fluorophore species that are excited with a single or two different laser lines and detected in two non-overlapping spectral channels. However, signaling pathways in biological systems often involve interactions between multiple biomolecules, e.g. formation of ternary or quaternary protein complexes. Here, we present a methodology to investigate such interactions at the plasma membrane (PM) of cells, as encountered for example in viral assembly or receptor-ligand interactions. To this aim, we introduce scanning fluorescence spectral correlation spectroscopy (SFSCS), a combination of scanning fluorescence correlation spectroscopy with spectrally resolved detection and decomposition. We first demonstrate that SFSCS allows cross-talk-free cross-correlation analysis of PM-associated proteins labeled with strongly overlapping fluorescent proteins (FPs), such as mEGFP and mEYFP, excited with a single excitation line. We then verify the applicability of SFSCS for quantifying diffusion dynamics and protein oligomerization (based on molecular brightness analysis) of two protein species tagged with spectrally overlapping FPs. Adding a second laser line, we demonstrate the possibility of three- and four-species (cross-) correlation analysis using mApple and mCherry2, as examples of strongly overlapping FP tags in the red spectral region. Next, we apply this scheme to investigate the interactions of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors simultaneously. Using the same set of fluorophores, we furthermore extend the recently presented raster spectral image correlation spectroscopy (RSICS) approach to four species analysis, successfully demonstrating multiplexed RSICS measurements of protein interactions in the cell cytoplasm. Finally, we apply RSICS to investigate the assembly of the ternary IAV polymerase complex and report a 2:2:2 stoichiometry of these protein assemblies in the nucleus of living cells.

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“…Fluorescence correlation spectroscopy (FCS) reveals differences in the dynamics of wildtype and mutant β-catenin FCS is a powerful quantitative microscopy method for measuring local concentrations and diffusional mobilities of fluorescent molecules in cells and tissues, e.g., to determine Wnt ligandreceptor interactions in the cell membrane (Eckert et al, 2020). To simultaneously monitor the dynamics of wild-type Clover-tagged and mutant Cherry-tagged β-catenin of HCT116 cells clone #37, we used FCS with detection in two color channels.…”

Section: Wild-type and Mutant β-Catenin Variants Contribute Both To Cmentioning

confidence: 99%

“…For FCS analysis, intensity time traces of 120 s duration were acquired in two color channels for 120 s each by using a home-built confocal microscope described previously (Eckert et al, 2020). Clover and Cherry were excited with pulsed 470-and 561-nm laser light of typically 3 µW (1.5 kW/cm 2 ) and 5 µW (2.8 kW/cm 2 ), respectively.…”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

“…Two correction factors were applied to the correlation amplitudes of each individual cell measurement to account for photobleaching and uncorrelated background due to cellular autofluorescence. The correction for photobleaching was described recently (Eckert et al, 2020). To correct for autofluorescence according to (Foo et al, 2012), we measured background intensities in the cytosol and in the nucleus for ensembles of more than 15 cells of lines #45 and #33, both under control conditions (DMSO) and with drug treatment (CHIR99021).…”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

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

Ambrosi

Voloshanenko

Eckert

et al. 2020

Preprint

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Wnt signaling plays important roles in embryonic development and during tissue homeostasis, and its deregulation contributes to the onset and progression of tumors. β-catenin is a key component of the 'canonical' Wnt pathway. Upon activation of the pathway, β-catenin is stabilized and enters the nucleus to bind transcription factors and activate Wnt target genes. Mutations that activate β-catenin have been found in many gastrointestinal tumors, including colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we engineered fluorescently tagged alleles of the endogenous βcatenin by CRISPR/Cas9 in a colorectal cancer cell line. We generated a cell model which carries wild-type and the oncogenic 45 mutant allele tagged with different fluorescent proteins, enabling the functional and biochemical analysis of both variants in the same cell. Endogenously tagged β-catenin remains functional, responds to Wnt pathway activation and does not interfere with the role of β-catenin in cell-cell adhesion. We then analyzed their biochemical and biophysical properties using immunoprecipitation, immunofluorescence and fluorescence correlation spectroscopy approaches. Our results show that wild-type and mutant β-catenin have distinct biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele found in colorectal cancer. The one-step tagging strategy also demonstrates how CRISPR/Cas9-mediated genome engineering can be employed for the concurrent functional analysis of wild-type and mutant alleles.

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

Cited by 28 publications

References 45 publications

Fast Diffusion Sustains Plasma Membrane Accumulation of Phosphatase of Regenerating Liver-1

Fast Diffusion Sustains Plasma Membrane Accumulation of Phosphatase of Regenerating Liver-1

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

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

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