Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Summary

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

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.

Introduction

Förster Resonance Energy Transfer (FRET) is widely used to gain a better understanding of cellular functions in living cells with high temporal and spatial resolution¹. In FRET, energy from an excited donor fluorophore is transferred to an acceptor fluorophore. FRET efficiency is strongly dependent on the distance between the donor and acceptor fluorophore and their orientation and is therefore a sensitive readout of conformational changes that affect the two fluorophores. This phenomenon is exploited to generate FRET-based biosensors for the imaging of small molecules. Changes in their concentration can be monitored as increases/decreases in the ratio....

Protocol

1. Transient Transfection

Note: CytoBAS and NucleoBAS (Please see Materials Table) are successfully used in several cell types, (U2OS, Huh7, HepG2, H69, MDCK and HEK293T cells). The main requirement to use the sensor is that it needs to be expressed, requiring the encoding DNA to enter the cell.

1. Harvest cells from an 80% confluent 25 cm² flask. Dilute cells in complete culture medium suitable for the specific cell line (10% FBS, 1% L-glutamine, 1% pen/strep for U2OS and Huh7 cells).
2. Plate cells at the desired density into a sterile 8 well chambered cover glass (0.8 cm²). Aim for a sub-confluent (6....

Results

The FRET-BAS sensor presented is based on the ligand binding domain of FXR (LBD-FXR) attached to two fluorophores citrine and cerulean) and an LXXLL motif. This sensor allows investigations into bile acid transport in living cells with high spatial and temporal resolution (Figure 1A). Mutations in cerulean and citrine were applied to promote the formation of the intramolecular complex (Figure 1B

Discussion

Here we present a detailed protocol for the use of a novel genetically encoded bile acid sensor capable of monitoring the spatiotemporal dynamics of bile acid transport in living cells. This biosensor consists of cerulean and citrine fluorescent proteins that are fused to FXR-LBD, thereby forming a FRET-based bile acid sensor (BAS).

The Bile Acid Sensor is relatively simple and convenient in use when having basic experience with cell culture and FACS or (confocal) microscopy. However, some asp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CytoBAS	Addgene	62860
NucleoBAS	Addgene	62861
Dulbecco's modified Eagles media (DMEM)	Lonza	BE12-614F	High glucose without L-glutamine
Penicillin-Streptomycin (pen/strep)	Lonza	17-602E
L-glutamine (200mM)	Lonza	17-605E
Fetal Bovine Serum (FBS)	Invitrogen	102-70
Trypsin-EDTA (10x)	Lonza	CC-5012
T-25 cell culture flask	VWR international	392-0253	Laminin coated
T-175 cell culture flask	VWR international	392-0238	Laminin coated
6-well plate	VWR international	734-0229	Poly-L-lysine and Laminin coated
10 cm dish	VWR international	392-0243	Laminin coated
Diethylaminoethyl (DEAE) - Dextran	Sigma-Aldrich	D9885
Polyethylenimine (PEI)	Brunschwig	23966-2
G418 (geneticin) 50 mg/ml	Invitrogen	10131-027
Hygromycin B, 50 mg/ml	Invitrogen	10687-010
Cloning cylinder (6 x 8 mm)	Bellco	2090-00608
L-15 Leibovitz culture medium	Invitrogen	21083-027	No phenol red
Polystyrene round bottom tube (5 ml) Facs tube	Falcon BD	352008	No cap, non-sterile
Falcon 2,063 tubes (5 ml)	Falcon BD	352063	Snap cap, sterile
Nunc Lab-Tek 8 well coverglass	Thermo scientific	155409	Sterile
Charcoal-filtered FBS	Life technologies	12676011
GW4064	Sigma-Aldrich	G5172
TCDCA	Sigma-Aldrich	T6260
CDCA	Sigma-Aldrich	C9377
Other chemicals	Sigma-Aldrich	n.v.t.