title: Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor.
abstract: Förster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Academic Medical Center.
authors.forename: Sandra
authors.initials: S
authors.lastname: Van de Wiel
authors.affiliation: Laboratory of Chemical Biology, Institute of Complex Molecular Systems (ICMS); Department of Biomedical Engineering, Eindhoven University of Technology.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Academic Medical Center; k.f.vandegraaf@amc.uva.nl.
authors.forename: Stan
authors.initials: S
authors.lastname: Van de Graaf
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003199:Computer Systems; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal / Q000379:methods; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D001647:Bile Acids and Salts; D005456:Fluorescent Dyes; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D000099150:Receptor, Farnesoid X-Activated