Name: real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor
Uploaded: 2016-01-04T15:00:01
Description: Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

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).

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.
<ol>
	<li>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 ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+imaging.">FRET imaging.</a> Nature Biotechnol. 21 (11), 1387-1395 (2003).</li><li>Mank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys. J. 90 (5), 1790-1796 (2006).</li><li>Li, I. T., Pham, E., Truong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+the+principle+of+fluorescence+resonance+energy+transfer+for+monitoring+cellular+dynamics.">Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics.</a> J. Biotechnol. Lett. 28 (24), 1971-1982 (2006).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li><li>Merkx, M., Golynskiy, M. V., Lindenburg, L. H., Vinkenborg, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+FRET+sensor+proteins+based+on+mutually+exclusive+domain+interactions.">Rational design of FRET sensor proteins based on mutually exclusive domain interactions.</a> Biochem. Soc. Trans. 41 (5), 1201-1205 (2013).</li><li>Lindenburg, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Stickiness:+Thermodynamic+Characterization+of+Intramolecular+Domain+Interactions+To+Guide+the+Design+of+Fo&#776;rster+Resonance+Energy+Transfer+Sensors.">Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of Fo&#776;rster Resonance Energy Transfer Sensors.</a> Biochem. 53 (40), 6370-6381 (2014).</li><li>van der Velden, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+bile+acid+transport+in+single+living+cells+using+a+genetically+encoded+F&#246;rster+resonance+energy+transfer+sensor.">Monitoring bile acid transport in single living cells using a genetically encoded F&#246;rster resonance energy transfer sensor.</a> J. Hepatol. 57 (2), 740-752 (2013).</li><li>Dawson, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heteromeric+organic+solute+transporter+&#945;-&#946;,+Ost&#945;-Ost&#946;,+is+an+ileal+basolateral+bile+acid+transporter.">The heteromeric organic solute transporter &#945;-&#946;, Ost&#945;-Ost&#946;, is an ileal basolateral bile acid transporter.</a> J. Biol. Chem. 280 (8), 6960-6968 (2005).</li><li>Meier, P. J., Stieger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salt+transporters.">Bile salt transporters.</a> Annu. Rev. Physiol. 64 (1), 635-661 (2002).</li><li>Klarenbeek, J. B., Goedhart, J., Hink, M. A., Gadella, T. W., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mTurquoise-based+cAMP+sensor+for+both+FLIM+and+ratiometric+read-out+has+improved+dynamic+range.">A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.</a> PLoS One. 6 (4), e19170 (2011).</li><li>Wallrabe, H., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+molecules+using+FRET+and+FLIM+microscopy.">Imaging protein molecules using FRET and FLIM microscopy.</a> Curr. Opin Biotechnol. 16 (1), 19-27 (2005).</li><li>Plass, J. 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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...

F&#246;rster 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 (OST&#945;&#946;)8. NTCP is the principal hepatic bile acid importer and OST&#945;&#946; 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 OST&#945;&#946;, 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.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CytoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right" style="width:145px;">62860</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoBAS&#160;</td>
			<td style="width:173px;">Addgene</td>
			<td align="right">62861</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Dulbecco&#39;s modified Eagles media (DMEM)</td>
			<td style="width:173px;">Lonza</td>
			<td>BE12-614F</td>
			<td>High glucose without L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin (pen/strep)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-602E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-glutamine (200mM)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">102-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Trypsin-EDTA (10x)</td>
			<td style="width:173px;">Lonza</td>
			<td style="width:145px;">CC-5012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-25 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0253</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T-175 cell culture flask</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0238</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">734-0229</td>
			<td>Poly-L-lysine and Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">10cm dish</td>
			<td style="width:173px;">VWR international</td>
			<td style="width:145px;">392-0243</td>
			<td>Laminin coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Diethylaminoethyl (DEAE) - Dextran</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">D9885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Polyethylenimine (PEI)&#160;</td>
			<td style="width:173px;">Brunschwig</td>
			<td style="width:145px;">23966-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">G418 (geneticin) 50 mg/ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10131-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Hygromycin B, 50 mg / ml</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">10687-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cloning cylinder (6x8 mm)</td>
			<td style="width:173px;">Bellco</td>
			<td style="width:145px;">2090-00608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">L-15 Leibovitz culture medium</td>
			<td style="width:173px;">Invitrogen</td>
			<td style="width:145px;">21083-027</td>
			<td>No phenol red</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Polystyrene round bottom tube (5 ml) Facs tube</td>
			<td style="width:173px;">Falcon BD</td>
			<td style="width:145px;">352008</td>
			<td>No cap, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Falcon 2063 tubes (5 ml)</td>
			<td style="width:173px;">Falcon BD</td>
			<td align="right" style="width:145px;">352063</td>
			<td>Snap cap, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Nunc Lab-Tek 8 well coverglass</td>
			<td style="width:173px;">Thermo scientific</td>
			<td style="width:145px;">155409</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Charcoal-filtered FBS</td>
			<td style="width:173px;">Life technologies</td>
			<td style="width:145px;">12676011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">GW4064</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">G5172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">T6260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">CDCA</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">C9377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Other chemicals</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:145px;">n.v.t.</td>
			<td></td>
		</tr>
	</tbody>
</table>

real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor

F&#246;rster 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.

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</...

Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

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

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.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

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