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 of emission intensity of the acceptor versus the donor fluorophore². For instance, FRET-based calcium biosensors allow for fast and stable detection of free calcium concentrations in living cells³. Other advantages of FRET-based biosensors are imaging in single living cells, their non-invasiveness, their ability to be targeted to different cell types and cellular compartments⁴.

Many aspects of intracellular bile acid dynamics are still poorly understood. For example, little is known about the mechanism underlying regulation of conjugated and unconjugated bile acid transport. Existing techniques to monitor this transport primarily make use of luciferase-based reporters, radiolabeled bile acids, or fluorescent bile acid analogs. The latter requires modification of bile acids, possibly affecting their properties. Luciferase-based reporters have poor time resolution. Besides, these techniques result in loss of the sample and are not applicable for imaging in single cells. Therefore, it would be beneficial to use methods that allow live single cell imaging of transport activity using FRET biosensors, especially since it includes the advantage of ratiometric detection^5,6. While variants of CFP/YFP form most frequently used FRET pairs, new strategies using mOrange and mCherry carrying self-association-inducing mutations have led to an expansion of the FRET toolbox with novel sensors, including a red-shifted bile acid sensor⁷.

We previously created a genetically-encoded FRET bile acid sensor (BAS), that consists of a donor fluorophore (cerulean) and an acceptor fluorophore (citrine) that are fused with the farnesoid X receptor (FXR) ligand binding domain (FXR-LBD) and a peptide containing an LXXLL motif⁸. This peptide associates with the FXR-LBD in a bile acid-dependent manner. Upon FXR activation, the distance between citrine and cerulean will alter due to a conformational change. In mammalian cell lines, FXR activation results in a clearly detectable increase in the citrine/cerulean ratio, while the purified sensor works in the opposite direction and leads to a decreased FRET ratio upon FXR activation. This sensor (CytoBAS) allows monitoring of cytosolic bile acid dynamics. By carboxyl-terminal addition of subcellular targeting motifs, the BAS construct can be targeted to the nucleus (NucleoBAS) and peroxisomes (PeroxiBAS), allowing measurements of bile acid concentrations in different cellular compartments. Although the addition of the peroxisomal targeting motif does not impair its responsiveness to bile acids, cell permeable FXR-ligands did not induce any FRET changes of PeroxiBAS inside peroxisomes⁸. As the nature of this discrepancy is unknown, the protocol below is focused on CytoBAS and NucleoBAS.

The use of this genetically encoded FRET sensor was recently demonstrated in cells containing the hepatic bile acid transporters Na⁺/taurocholate co-transporting polypeptide (NTCP) and organic solute transporter alpha / beta (OSTαβ)⁸. NTCP is the principal hepatic bile acid importer and OSTαβ is a basolateral intestinal bile acid transporter that can function both as an importer and exporter dependent on the electrochemical bile acid concentration gradient^9,10. Recent data showed that upon bile acid transport by NTCP and/or OSTαβ, robust and fast responses in FRET ratio as a result of ligand-FXR-LBD interaction can be observed.

Here, we describe detailed protocols for methods to measure FRET such as confocal microscopic analysis and fluorescence activated cell sorting (FACS), highlight critical steps, address potential problems and discuss alternative methods. Using this genetically encoded FRET sensor, bile acid interaction with FXR-LBD can be quantified and monitored directly in living cells and provides a rapid and simple method of visualizing bile acid transport and dynamics in real-time. Mammalian expression plasmids encoding CytoBAS and NucleoBAS are available commercially. Therefore, this biosensor can further contribute to the understanding of bile acid transporters or compounds that activate FXR and provide a deeper insight into bile acid biology and signaling.

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 (60-80% confluency) layer of cells on the day of the experiment, although this is not essential.
3. Add complete culture medium to a final volume of 400 µl per well. Allow cells to attach for 24 hr.
4. Prepare the transfection mix in a sterile tube by adding 0.5 µg of NucleoBAS or CytoBAS DNA to 100 µl of culture medium (serum/antibiotic free). Vortex well. Add a transfection reagent and mix by vortexing. Transfection reagents that give optimal efficiency are cell type dependent and might require prior testing.
   1. For instance, use 2.5 µl of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.
5. Add the transfection mixture in droplets to the cells and mix by gently shaking the well plate by hand. Add 100 µl of the mixture for a well of ~9.6 cm². Use 10 µl of the transfection mixture for individual imaging chambers for transiently transfected cells.
   Note: After 24-48 hr, cells will express the bile acid sensor and can be used for experiments.

2. Stable Transfection

1. Harvest cells from an 80% confluent 25 cm² flask. Dilute pellet in complete culture medium suitable for the specific cell line (10% Fetal Bovine Serum, 1% L-glutamine, 1% pen/strep for U2OS and Huh7 cells) to a concentration of 150,000 cells per ml and add around 300,000 cells per well in a 6-well culture plate (2 ml end volume).
2. Allow cells to attach for 24 hr.
3. Prepare the transfection mix in a sterile tube by adding 0.5 µg of CytoBAS or NucleoBAS DNA (Please see Materials Table) to 100 µl of culture medium (serum/antibiotic free). Vortex well. Add a transfection reagent and mix by vortexing.
   1. For instance, use 2.5 µl of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.
4. Add 100 µl of the transfection mixture in droplets to the cells and mix by gently shaking the well plate by hand. Always include an untransfected control when creating a stable cell line.
5. Grow cells O/N at 37 °C, 5% CO₂.
6. Trypsinize cells according to manufacturer's protocol (Incubate cells at 37 °C with 1.5 ml prewarmed trypsin per 25 cm² cell culture) and spin them down at 250 x g at RT for 5 min. Remove supernatant and resuspend cells in 13 ml of complete culture medium. Divide cells over various 10 cm diameter culture dishes: 0.5 ml (low density), 1.5 ml (medium density), and 4.5 ml (high density). Do the same for remaining 6.5 ml.
7. Add culture medium to a final volume of 10 ml. For U2OS cells, use 800 µg/ml G418 for plasmids with the neomycin resistance cassette such as the CytoBAS and NucleoBAS constructs to select for positive cells. This concentrations is cell type dependent and can be determined by selecting the lowest antibiotic (G418) concentration where untransfected cells died within 2 to 10 days (800 µg/ml G418 for U2OS cells).
8. Refresh the culture medium with the appropriate antibiotic (G418) every 72 hr until the untransfected cells are dead.
9. Examine the culture plate under the microscope with a 20X objective for positive colonies (fluorescence can be monitored using most filter sets used for green, cyan or yellow fluorescence).
10. Mark the positive colonies that lie isolated from other colonies with a pen on the exterior bottom of the culture dish.
11. Wash the plate twice with Phosphate Buffered Saline (PBS) and aspirate it. Take around 15 sterile cloning cylinders and dip them into sterile silicone. Apply them around the selected colonies by pressing lightly against the petri-dish and make sure that not more than one colony is included in the cloning ring.
12. Pipet 20 µl of pre-warmed trypsin (1x, 0.25%) in the cloning cylinder and incubate at 37 °C until most of the cells have rounded up (observe with a microscope).
13. Add 150 µl of culture medium with serum and the appropriate antibiotic (10% FBS and 800 µg/ml G418 for U2OS and Huh7 cells) in the cloning cylinder and pipet up and down several times to resuspend the cells and transfer to a 96-well plate. Refresh medium after 4-24 hr and allow cells to grow confluent in the next few days (37 °C, 5% CO₂for U2OS and Huh7 cells).
14. Refresh medium with antibiotic every 3 or 4 days. When the cells have grown confluent, transfer them to a larger well plate. Repeat this step until the culture is large enough for experiments. Cryopreserve some vials for backup. Cryopreservation medium consists of 20% FBS and 20% DMSO in media (DMEM) without supplements.
    Note: Now, the cell line is monoclonal and considered stable for expressing the Bile Acid Sensor. Half the concentration of the antibiotic G418 (400 µg/ml) is now sufficient to maintain expression in the stable cell line.

3. Lentiviral Transduction

Note: Some cell lines are considered difficult to transfect by more traditional methods such as the polyethylenimine (PEI) method. Viral transduction of cells is an efficient alternative tool for gene-delivery and stable transgene expression.

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) and add around 200,000 cells per well in a 6-well culture plate.
2. Add culture medium to a final volume of 2 ml per well. Allow cells to attach for 24 hr.
3. Perform viral transductions by incubating cells for 4-6 hr with 500 µl CytoBAS lentivirus containing medium (available upon request) filled up to a total amount of 1 ml with complete culture medium containing 10 µg/ml diethylaminoethyl (DEAE)-dextran or another lentiviral transduction enhancer. Do not forget to include a well with untransduced control cells.
4. Remove medium and add 2 ml of complete culture medium containing the desired antibiotic (500 µg/ml hygromycin). Use a lentiviral construct for CytoBAS transduction that provides hygromycin resistance to cells. Grow cells under desired conditions.
5. Refresh medium with (500 µg/ml hygromycin) antibiotic every 3 days and split the cells when 80% confluent, until all negative control cells are dead (takes about 1-2 weeks for most cell lines). The cell line is now considered stable for expressing the Bile Acid Sensor.
   1. Alternatively, use cells for experiments 1-3 days after viral transduction. As live virus might be present, this requires FRET-readout equipment in rooms with the appropriate safety level.
6. Alternatively, for rapid isolation of stable cell lines, perform fluorescence activated cell sorting (FACS).
   1. Harvest cells from a confluent 160 cm² flask.
   2. Dilute cells in Leibovitz culture medium (L-15 medium) with <5% serum to a concentration of maximum 5 x 10⁶ cells per ml
   3. For collection of sorted cells, fill FACS tubes with 1 ml of collection medium (L-15 medium + 1.5% pen/strep, 1% L-glut and 20% serum).
   4. Sort cells for citrine (520-580 nm) or cerulean (450-520 nm), excited with 405 laser.
   5. After sorting approximately 500,000 cells, spin down cells (5 min, 250 x g) and resuspend them in 5 ml normal complete culture medium (10% FBS, 250 µg/ml Hygromycin for U2OS and Huh7 CytoBAS cells) and plate them in a T25 culture flask. Grow cells under desired conditions (37 °C, 5% CO₂ for U2OS and Huh7 cells).

4. Live Cell Imaging of the Bile Acid Sensor

Note: Cells containing bile acid transporters can be cultured in medium with 1-10% charcoal-filtered serum that removes lipophilic compounds. Normal serum often contains bile acids that could lead to intracellular bile acid accumulation and saturation of the Bile Acid Sensor.

1. FRET measurements using the confocal microscope
   1. Plate the cells stably or transiently expressing the sensor at the desired density into a sterile 8 well coverslip bottomed chamber slide (0.8 cm²). Grow cells in complete culture medium (using charcoal-filtered serum) so that on the day of the experiment, confluency is around 60-70%.
   2. Wash the adherent cells in chamber slide once with 200 µl 1x PBS or Leibovitz's L-15 culture medium.
   3. Aspirate and replace with 300 µl Leibovitz's L-15 culture medium so that no CO₂control is necessary.
      Note: DMEM without phenol red can be used as well but requires CO₂buffered conditions. The bile acid sensor shows (some) pH-sensitivity so aim to keep the pH constant during the data-collection.
   4. Dilute the compounds to be used in the confocal imaging experiment also in L-15 culture medium. Take into account that during imaging, relatively large amounts of liquid in the chambers have to be added to secure rapid mixing of the sample (50-100 µl), so make 3-5x solutions.
   5. Start the imaging software of a confocal microscope with a 37 °C incubation chamber and turn on the violet 405 nm laser. Put a drop of immersion oil onto the objective and place the 8-chamber on top of it. For single cell imaging of FRET, use the 63X oil objective. The sensor has been successfully used at RT, but cell behavior might be altered.
   6. Set the settings of the confocal microscope properly.
      1. Set Acquisition mode: xyt (time-lapse imaging of single focal plane).
      2. Acquire images at 20 sec intervals to allow compounds to be added between acquisitions without pausing the experiment.
      3. Set spectral range for emission detection: Cerulean: 450-520 nm; Citrine: 520-580 nm.
   7. Focus on the single cell layer using transillumination light. Start imaging and adjust the z-position more precisely. Adjust gain and offset for each channel to distinguish signal from background while remaining well below saturation for all pixels in cells of interest.
   8. Draw circles to define the region of interest (ROI). Select cells that show similar fluorescence intensity. Avoid cells that obviously differ from the average cell in size and shape. Expose the cells to light for a time as short as possible to minimize photobleaching of the sensor. It is advisable to draw ROIs not very close to the cell perimeter. This area is most sensitive to changes in fluorescence intensity due to focal drift (cells moving in z-direction) making it more difficult to monitor changes in fluorescence ratio during the experiment.
   9. Start measurements with the confocal microscope. Wait until the cerulean and citrine fluorescence is stable.
   10. Add 50-100 µl bile acids or other compounds at chosen time points during measuring. The relatively high amounts of liquid (one-fifth to one-third of the final volume) are necessary to mix the fluids well (within 10 sec) without shaking/pipetting up and down. Make sure not to touch the edge of the 8-well chamber with the pipette tip and add the liquid slowly so that the cells do not get out of focus. Do not add new compounds before the plateau phase is reached. This takes about 200 sec.
   11. End each experiment with the addition of 100 µl GW4064, end concentration 5-10 µM (sensor saturating dose) and wait until sensor fluorescence is stable.
   12. Save the experiment and export the data as an .AVI file.
   13. Open in ImageJ both AVI files (channel 00, cerulean; channel 01, citrine) by selecting Plugins > Stacks > Stack interleaver. Alternatively, import confocal file (e.g., .lsm or .lif files) directly in Image J using appropriate plugins (available at http://www.openmicroscopy.org) and move to step 4.1.14.
       1. For Stack 1: use Channel 01 (citrine).
       2. For Stack 2: use Channel 00 (cerulean).
   14. Click on Edit > Selection > Add to manager, to open the ROI manager window. Check the checkbox 'Show All'.
   15. Draw a few regions of interest (ROIs) covering specific cells with the oval selection tool. Also draw one circle in an area outside cells or inside a cell that does not express the sensor to determine the background signal. It is advisable to draw ROIs not very close to the cell perimeter in experiments when changes in fluorescence intensity due to focal drift (cells moving in z-direction) or cell migration were obvious.
   16. Select one cell. Click on Plugins > Ratio Profiler. This will result in 3 screens: RAW, ratio and Ratio_Profile. The RAW window shows the increase in intensity of citrine (blue line) and a decrease in cerulean (red line) if there is FRET. The Ratio window gives information about the ratio citrine/cerulean, which will increase with an increase in FRET. The Ratio_Profile window gives the actual numbers of fluorescence intensity measured in both channels. If microscope setup-specific files (e.g., .lsm or .lif instead of .avi) files are used the channel order might be reversed.
   17. Copy the data from the Ratio_Profile window in the spreadsheet attached as supplementary data. Do the same for all the other cells (and background ROI).
       Note: In the online spreadsheet, all data is normalized to the condition at which maximum BAS activation is expected. Given that GW4064 is the most potent activator of FXR, the fluorescence ratio after incubation with a surplus of GW4064 is set to 1. It is therefore important to end all of the experiments with addition of GW4064. The advantage of this is that the data is no longer dependent on laser intensity or detector gain and experiments on different days can be compared more easily. Furthermore, in the bottom graph of the spreadsheet, a running average can be used to smooth the curves for experimental noise. However, do not use this graph when analyzing kinetic data, since the running average will also smooth fast kinetic responses.
2. FRET measurements using Fluorescence Activated Cell Sorting (FACS)
   1. Dilute all compounds for the FACS experiment in sterile FACS uptake buffer (0.3 mM EDTA, 0.5% BSA, 0.01% NaN₃and 10 mM D-glucose).
   2. Harvest cells from an 80% confluent T-160 cm² cell culture flask using 5 mM EDTA in PBS. Centrifuge cells at 250 x g for 5 min. Wash cell pellet 2x in 5 ml FACS uptake buffer at RT.
   3. Count cells using the coulter counter or a counting chamber.
   4. Dilute pellet in FACS uptake buffer to a concentration of 1 x 10⁶ cells/ml. Pipette up and down to create a homogeneous suspension of single cells. If cells are difficult to disaggregate, put the samples through a cell strainer before sorting to minimize nozzle clogs.
   5. Pipet 200 µl cells per FACS tube and protect them from light.
   6. Add the desired concentration of the compound (e.g., bile acids, synthetic FXR ligands, transporter inhibitors). Vortex. Incubate for 20-30 min at RT while shaking (in the dark).
   7. Meanwhile, start the FACS (the lasers need time to warm up).
   8. Set the flow cytometry gating parameters for the experiment (see Figure 3):
      1. Load around 100,000-200,000 NucleoBAS or CytoBAS transfected cells to determine the gates.
      2. First adjust the FSC and SSC voltages to plot the cells in the center of the plot.
      3. Using the violet laser, adjust the cerulean (450/40 nm) voltage value and citrine (525/20 nm) voltage and ensure that all NucleoBAS or CytoBAS positive cells are plotted within the scatter plot.
      4. Set the correct gates (Gate P1 up to Gate P4), see Figure 4.
         1. Use gate P1 to exclude dead cells, usually displayed in the lower left corner by selecting the main population in the middle of the SSC-A/FSC-A plot.
         2. Use gate P2 in the FSC-H/FSC-A window to remove duplets from analysis. Single cells are presented in a more diagonal line than doublets.
         3. Gate P3 in the citrine (525/20 nm)/cerulean (450/40 nm) window can be used to select for NucleoBAS/CytoBAS positive cells, by gating citrine and cerulean high cells, a population displayed diagonally in the upper right corner of the plot.
         4. Draw gate P4 at the top left of the population, as close as possible and make sure that no more than 5% of the cells fall within this gate.
      5. Load some untransfected cells (no expression of CytoBAS or NucleoBAS) to determine auto-fluorescence. The untransfected population may not be located in gate P3. Adjust gate P3 when necessary to exclude auto-fluorescent cells.
   9. Vortex the samples before placing the tubes in the FACS. For each sample, measure at least 10,000 cells in gate P3.
      Note: The Population Hierarchy Window gives the number of events being displayed for each gate and % of the parent gate. In gate 4, the % of the parent states cells with an increased citrine/cerulean ratio as a percentage of all NucleoBAS positive cells.

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.