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.
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.
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 detection5,6. 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 gradient9,10. 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.
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.
2. Stable Transfection
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.
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.
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). Bile acids and other FXR ligands bind to FXR and thereby alter the distance between citrine and cerulean by a conformational change. In living mammalian cells, this results in an increased FRET efficiency (citrine increase, cerulean decrease) (Figure 1C, 1D). During a live cell quantitative intensity-based FRET experiment with the confocal microscope, it was observed that taurochenodeoxycholic acid (TCDCA)-treated (30 µM) U2OS cells expressing NucleoBAS lacking bile acid transporters do not show any changes in FRET, while the average citrine/cerulean ratio increased remarkably after treatment with GW4064 (5 µM) (Figure 1C). In contrast, U2OS cells co-expressing NucleoBAS, NTCP and OSTαβ did show a clear increase in citrine/cerulean ratio upon addition of TCDCA, and an even larger increase in ratio after addition of GW4064 (Figure 1D). This demonstrates that TCDCA is unable to pass through the cell membrane and requires specific transporters to enter the cell.
Cellular localization of the sensor is determined by the presence of specific localization signals. NucleoBAS is targeted to the nucleus by the nuclear localization signal (Figure 2B), while CytoBAS does not contain any localization signal and therefore remains in the cytosol (Figure 2A). The biosensor can also be combined with imaging of other fluorescent proteins, enabling measurements of protein expression/localization and FXR activation in the same cell. For instance, Figure 2C shows U2OS cells co-transfected with NucleoBAS and NTCP-mKate2. Additionally, the sensor can also be combined with other sensors for subcellular imaging of more than one parameter simultaneously. For instance, NucleoBAS was expressed along with TGR5 (EST clone IMAGE #5221127) and a cAMP cytosolic sensor (TEpacVV)11 to measure TGR5 and FXR activation in the same cell at the same time. Upon TGR5 binding, cAMP levels increased in the cytosol which was detected by the cAMP sensor, while FXR activation in the nucleus was visualized simultaneously. Figures 2D-F show a time series composed of 6 confocal images of NucleoBAS-TGR5-TEpacvv cells after treatment with GW4064 (5 µM) (Figure 2D), with TCDCA (10 µM) (Figure 2E) or with chenodeoxycholic acid (CDCA) (10 µM) (Figure 2F) at t = 0. The green color represents regions where 525/450 nm emission ratio is decreased (representing cAMP elevation) and the pink color represents an increase in emission ratio (FXR activation), compared to the ratios at the start of the experiment.
In addition, flow cytometry can be used to screen a pool of cells on single cell level or as high-throughput screening on the whole population. U2OS cells expressing NucleoBAS were excited with a violet 405 nm laser and the fluorescence was collected in the 450/40 range for cerulean and 525/20 range for citrine. In order to improve the efficiency of FACS-based FRET experiments, proper gates must be set (Figure 3). The P1 gate excludes cell debris and the P2 gate removes duplets from analysis. Next, the analysis is restricted to citrine (excited with 488 laser) and cerulean (excited with 405 laser) positive cells by gate 3. Finally, gate P4 represents the percentage of cells with a high citrine/cerulean emission ratio (using excitation of cerulean with 405 nm laser). For each sample, at least 10,000 cells that fell within gate 3 were measured.
Subsequently, FACS-based FRET flow cytometry was used to calculate an increase in FRET in living cells after 30 min incubation with TCDCA and GW4064. Non-treated U2OS cells expressing NucleoBAS showed <5% cells in gate P4 (citrine/cerulean-high cells). In absence of any bile acid transporters, a similar percentage was observed in 30 µM TCDCA treated cells (Figure 4A). In contrast, cell co-expressing NucleoBAS, NTCP and OSTαβ did show an increased amount of citrine/cerulean-high cells of around 38%. After addition of 10 µM GW4064, almost 90% of the cells were citrine/cerulean-high irrespective of expression of OSTαβ or NTCP (Figure 4B). Data can be presented in bar graphs (% cells in gate 4, population 4) (Figure 4C, D) or as population histograms of citrine-cerulean ratio (Figure 4E, F).
Figure 1. Design of the Bile Acid Sensor (BAS). (A) Structural representation of the mode of action of a genetically encoded Bile Acid Sensor. It consists of a donor fluorophore (cerulean) and an acceptor fluorophore (citrine) linked via the FXR-LBD and fused to a FXR-cofactor peptide (LXXLL motif). (B) Schematic domain architecture of the BAS. The Q204F/V224L fluorophore mutations promote the formation of intramolecular interactions between cerulean and citrine. The NucleoBAS construct contains a c-terminal nuclear localization signal (NLS) and therefore accumulates in the nucleus, whereas the CytoBAS construct lacks any targeting sequence and therefore shows cytosolic localization. (C) Representative confocal-based FRET experiment with BAS as a tool to monitor conjugated bile acid transport. No change in citrine/cerulean ratio is observed after 30 µM TCDCA addition in cells only expressing NucleoBAS. When 5 µM GW4064 was added, a strongly increased citrine/cerulean ratio was observed. (D) Import of TCDCA (30 µM) results in a considerable activation of the sensor in NTCP and OSTαβ co-transfected cells. Subsequent GW4064 (5 µM) treatment leads to a further increase in citrine/cerulean ratio. Results are expressed as mean ± SD, n = 6 individual cells. Please click here to view a larger version of this figure.
Figure 2. Localization FRET-BAS construct in living cells. Confocal microscope images of U2OS cells transfected with CytoBAS (A) or NucleoBAS (B). (C) U2OS cells co-transfected with NucleoBAS and NTCP-mKate2. The scale bar represents 10 µm. (D–F) Time series of NucleoBAS-TGR5-TEPACVV transfected cells. (D) GW4064 activates FXR (increased 525/450 nm ratio), but not TGR5. (E) TCDCA activates TGR5 on the plasma membrane (decreased 525/450 nm ratio), but is not able to enter the cell to activate FXR in the nucleus. (F) CDCA activates TGR5 on the plasma membrane as well as FXR in the nucleus (increased 525/450 nm ratio in the nucleus, decreased in the cytoplasm). Please click here to view a larger version of this figure.
Figure 3. Flow cytometry gating parameters. (A) The P1 gate in the SSC-A/FSC-A window is used to exclude the dead cells. (B) In the FSC-H/FSC-A window, the P2 gate is drawn to eliminate doublet events from analysis. (C) In the citrine (525/20 nm)/cerulean (450/40 nm) window, NucleoBAS (and CytoBAS) positive cells are selected (Gate P3). (D) Finally, in the citrine (525/20 nm)/cerulean (450/40 nm) window, the P4 gate contains citrine/cerulean-high cells of the FACS experiment. Please click here to view a larger version of this figure.
Figure 4. Representative FACS experiment measuring bile acid transport using NucleoBAS. (A, B) FACS-plots showing the amount of citrine/cerulean-high living NucleoBAS-expressing U2OS cells (A) and U2OS cells co-expressing NucleoBAS, OSTαβ and NTCP (B) after treatment with control buffer (left), 30 µM TCDCA (middle) or 10 µM GW4064 (right). (C, D) Bar graph showing percentage of citrine/cerulean-high cells after 30 min incubation with TCDCA or GW4064 in U2OS cells expressing NucleoBAS and co-transfected with (D) or without (C) OSTαβ and NTCP. (E, F) Histogram showing the distribution of citrine/cerulean ratio in a population of U2OS cells expressing NucleoBAS with or without OSTαβ and NTCP after 30 min incubation with TCDCA (yellow), GW4064 (red) or control buffer (blue). All conditions were measured three times. Bars represent mean values + standard deviation (SD) (n = 3). Please click here to view a larger version of this figure.
Supplemental File 1: Template BAS general. Please click here to download this file.
Supplemental File 2: Template BAS Zeiss Leica imported data. Please click here to download this file.
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 aspects might require some trouble shooting. When using the bile acid sensor in combination with bile acid transporters, it is recommended to culture cells in medium with charcoal-filtered serum. Charcoal further reduces bile acids levels by adsorption from the serum. Especially in the presence of importers such as NTCP, this is crucial to prevent saturation of the sensor during culturing, since this is the most common cause of an insensitive sensor during experiments. Therefore, another sensor was created (NucleoBAS N354K/I372V) containing mutations altering the sensitivity for bile acids, creating a larger dynamic range8. To define whether the sensor is already saturated, the citrine/cerulean ratio can be compared to the ratio in control cells not expressing any bile acid transporters, since those cells are assumed to have low intracellular bile acid levels. Alternatively, fluorescence-lifetime imaging microscopy (FLIM) measurements can be performed to investigate FRET in an intensity-independent manner12. This technique is beyond the scope of this paper, and requires more specialized equipment. Other reasons for an insensitive sensor include the use of cells that are auto-fluorescent and/or have lost NucleoBAS expression. Of note, fluctuations in intensity of cerulean and citrine (for instance due to focal shifts) during the experiment can obscure direct visualization of FRET changes during imaging, while the ratiometric nature of the sensor still allows for monitoring of bile acid dynamics. Often absolute intensity changes are modest for the individual fluorophores upon addition of FXR ligands, while the increase in ratio is evident.
For FRET analysis in cells transfected with the BAS sensor, appropriate laser light and filters should be selected. The increase of citrine intensity during FRET has to be measured with the violet diode (405 nm) laser with emissions monitored over 450-520 (cerulean) and 520-580 (citrine). An important aspect to take into account is the sensitivity of the sensor to photobleaching. When the intensity of one or both fluorophores slowly declines in time without addition of ligands, it often indicates photobleaching. This phenomenon mainly occurs when the laser power is too high. To avoid this unwanted effect, the lasers power should not exceed 5% of the full power and the cells have to be kept in dark as much as possible. Limit the time to search for the right cells. Fluctuations in fluorescence intensity can also be caused by focal drift in the z-axis. To counteract this, the pinhole size can be increased and it is advisable to draw ROIs not very close to the cell perimeter.
The FRET sensor for bile acids has been tested in multiple cell types (U2OS, Huh7, HepG2, H69, MDCK and HEK293T cells) that can be transfected and have a stable phenotype. However, primary and differentiated cells such as hepatocytes are difficult to transfect and to maintain stable in terms of characteristics. Viral transduction can help with difficult-to-transfect cells (construct available on request). We are currently generating a mouse line to allow use of the sensor in freshly isolated primary cells that rapidly dedifferentiate upon isolation.
To perform the confocal experiment described, it is imperative that the selected cell line is adherent to a culture dish and grows in a single cell layer. However, cells that grow in suspension, or adherent cells that can be trypsinized rather easily, can also be measured using the FACS. Finally, it is advised to select a cell line without endogenous bile acid transport or synthesis when analyzing a specific transport pathway. i.e., when measuring the activity of certain transfected (mutant) transporter proteins.
The presented genetically encoded fluorescent biosensor BAS is a valuable tool for monitoring single live cell bile acid transport. The BAS contains a FXR-LBD which is activated by a large variety of bile acids, allowing imaging of subcellular dynamics of bile acids8. This gives a great advantage in relation to the use of other techniques. For instance, in promoter-driven luciferase reporter assays, the luciferase signal is dependent on the stability of the reporter within cells, does not support subcellular information, and requires the destruction of the sample13. Another approach to measure bile acid transport is the use of fluorescent labeled or radiolabeled ligands. However, the fluorescent labeling of bile acids can alter bile acid transport kinetics and the availability of radiolabeled bile acids is limited. Additionally, combining the sensor with expression of bile acid transporters, cellular influx and efflux of specific bile acids can be addressed. For instance, increased intracellular levels of conjugated bile acids like TCDCA provide insight about transport activity. This also makes it possible to use BAS to examine the effect on bile acid transport after interference with specific pathways, for instance by using molecular or compound inhibitor screens, or examining the effect of other FXR ligands. Finally, another application of the sensor is combining it with other fluorescent proteins for quantitative analysis or localization of proteins, for example imaging of plasma membrane transporters expression and FXR activation in the same cell.
The authors have nothing to disclose.
This work was supported by ERC starting grants (ERC-2011-StG 280255 and ERC-2013-StG 337479) and by the Netherlands Organization for Health Research and Development (Vidi 91713319).
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 |
10cm 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 (6×8 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 2063 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. |