Real time monitoring of intracellular bile acid dynamics using a genetically encoded FRET-based Bile Acid Sensor
| Autor: | Maarten Merkx, Stan F.J. van de Graaf, Sandra M. W. van de Wiel |
|---|---|
| Přispěvatelé: | Protein Engineering, Gastroenterology and Hepatology, Graduate School, Tytgat Institute for Liver and Intestinal Research, Amsterdam Neuroscience, Adult Psychiatry, Amsterdam Gastroenterology Endocrinology Metabolism |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cytoplasm medicine.drug_class General Chemical Engineering Receptors Cytoplasmic and Nuclear Bioengineering Biosensing Techniques Biology Ligands General Biochemistry Genetics and Molecular Biology Madin Darby Canine Kidney Cells Bile Acids and Salts 03 medical and health sciences Dogs Computer Systems Live cell imaging Fluorescence Resonance Energy Transfer medicine Animals Humans Cellular compartment Fluorescent Dyes Cell Nucleus Microscopy Confocal Bile acid General Immunology and Microbiology General Neuroscience Hep G2 Cells Cytosol HEK293 Cells 030104 developmental biology Förster resonance energy transfer Biochemistry Farnesoid X receptor Protein folding Intracellular |
| Zdroj: | Journal of Visualized Experiments (JoVE). MYJoVE Corporation ISSUE=107;ISSN=1940-087X;TITLE=Journal of Visualized Experiments (JoVE) Journal of visualized experiments. MYJoVE Corporation ISSUE=107;TITLE=Journal of visualized experiments |
| ISSN: | 1940-087X |
| DOI: | 10.3791/53659 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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