Name: real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor
Uploaded: 2016-01-04T15:00:01.000+00:00
Duration: 9 min 21 s
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 Huh7 cells).</li>
	<li>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.</li>
	<li>Add complete culture medium to a final volume of 400 &#181;l per well. Allow cells to attach for 24 hr.</li>
	<li>Prepare the transfection mix in a sterile tube by adding 0.5 &#181;g of NucleoBAS or CytoBAS DNA to 100 &#181;l 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.
	<ol>
		<li>For instance, use 2.5 &#181;l of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.</li>
	</ol>
	</li>
	<li>Add the transfection mixture in droplets to the cells and mix by gently shaking the well plate by hand. Add 100 &#181;l of the mixture for a well of ~9.6 cm2. Use 10 &#181;l 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.</li>
</ol>
2. Stable Transfection
<ol>
	<li>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).</li>
	<li>Allow cells to attach for 24 hr.</li>
	<li>Prepare the transfection mix in a sterile tube by adding 0.5 &#181;g of CytoBAS or NucleoBAS DNA (Please see Materials Table) to 100 &#181;l of culture medium (serum/antibiotic free). Vortex well. Add a transfection reagent and mix by vortexing.
	<ol>
		<li>For instance, use 2.5 &#181;l of 1 mg/ml polyethylenimine (PEI) and incubate the DNA/PEI mix for 15 min at RT.</li>
	</ol>
	</li>
	<li>Add 100 &#181;l 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.</li>
	<li>Grow cells O/N at 37 &#176;C, 5% CO2.</li>
	<li>Trypsinize cells according to manufacturer&#39;s protocol (Incubate cells at 37 &#176;C with 1.5 ml prewarmed trypsin per 25 cm2 cell culture) and spin them down at 250 x g at RT&#160;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.</li>
	<li>Add culture medium to a final volume of 10 ml. For U2OS cells, use 800 &#181;g/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 &#181;g/ml G418 for U2OS cells).</li>
	<li>Refresh the culture medium with the appropriate antibiotic (G418) every 72 hr until the untransfected cells are dead.</li>
	<li>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).</li>
	<li>Mark the positive colonies that lie isolated from other colonies with a pen on the exterior bottom of the culture dish.</li>
	<li>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.</li>
	<li>Pipet 20 &#181;l of pre-warmed trypsin (1x, 0.25%) in the cloning cylinder and incubate at 37 &#176;C until most of the cells have rounded up (observe with a microscope).</li>
	<li>Add 150 &#181;l of culture medium with serum and the appropriate antibiotic (10% FBS and 800 &#181;g/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 &#176;C, 5% CO2for U2OS and Huh7 cells).</li>
	<li>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 &#181;g/ml) is now sufficient to maintain expression in the stable cell line.</li>
</ol>
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.
<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 Huh7 cells) and add around 200,000 cells per well in a 6-well culture plate.</li>
	<li>Add culture medium to a final volume of 2 ml per well. Allow cells to attach for 24 hr.</li>
	<li>Perform viral transductions by incubating cells for 4-6 hr with 500 &#181;l CytoBAS lentivirus containing medium (available upon request) filled up to a total amount of 1 ml with complete culture medium containing 10 &#181;g/ml diethylaminoethyl (DEAE)-dextran or another lentiviral transduction enhancer. Do not forget to include a well with untransduced control cells.</li>
	<li>Remove medium and add 2 ml of complete culture medium containing the desired antibiotic (500 &#181;g/ml hygromycin). Use a lentiviral construct for CytoBAS transduction that provides hygromycin resistance to cells. Grow cells under desired conditions.</li>
	<li>Refresh medium with (500 &#181;g/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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Alternatively, for rapid isolation of stable cell lines, perform fluorescence activated cell sorting (FACS).
	<ol>
		<li>Harvest cells from a confluent 160 cm2 flask.</li>
		<li>Dilute cells in Leibovitz culture medium (L-15 medium) with &#60;5% serum to a concentration of maximum 5 x 106 cells per ml</li>
		<li>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).</li>
		<li>Sort cells for citrine (520-580 nm) or cerulean (450-520 nm), excited with 405 laser.</li>
		<li>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 &#181;g/ml Hygromycin for U2OS and Huh7 CytoBAS cells) and plate them in a T25 culture flask. Grow cells under desired conditions (37 &#176;C, 5% CO2 for U2OS and Huh7 cells).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>FRET measurements using the confocal microscope
	<ol>
		<li>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%.</li>
		<li>Wash the adherent cells in chamber slide once with 200 &#181;l 1x PBS or Leibovitz&#39;s L-15 culture medium.</li>
		<li>Aspirate and replace with 300 &#181;l Leibovitz&#39;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.</li>
		<li>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 &#181;l), so make 3-5x solutions.</li>
		<li>Start the imaging software of a confocal microscope with a 37 &#176;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.</li>
		<li>Set the settings of the confocal microscope properly.
		<ol>
			<li>Set Acquisition mode: xyt (time-lapse imaging of single focal plane).</li>
			<li>Acquire images at 20 sec intervals to allow compounds to be added between acquisitions without pausing the experiment.</li>
			<li>Set spectral range for emission detection: Cerulean: 450-520 nm; Citrine: 520-580 nm.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.</li>
		<li>Start measurements with the confocal microscope. Wait until the cerulean and citrine fluorescence is stable.</li>
		<li>Add 50-100 &#181;l 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.</li>
		<li>End each experiment with the addition of 100 &#181;l GW4064, end concentration 5-10 &#181;M (sensor saturating dose) and wait until sensor fluorescence is stable.</li>
		<li>Save the experiment and export the data as an .AVI file.</li>
		<li>Open in ImageJ both AVI files (channel 00, cerulean; channel 01, citrine) by selecting Plugins &#62; Stacks &#62; 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.
		<ol>
			<li>For Stack 1: use Channel 01 (citrine).</li>
			<li>For Stack 2: use Channel 00 (cerulean).</li>
		</ol>
		</li>
		<li>Click on Edit &#62; Selection &#62; Add to manager, to open the ROI manager window. Check the checkbox &#39;Show All&#39;.</li>
		<li>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.</li>
		<li>Select one cell. Click on Plugins &#62; 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>FRET measurements using Fluorescence Activated Cell Sorting (FACS)
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>Count cells using the coulter counter or a counting chamber.</li>
		<li>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.</li>
		<li>Pipet 200 &#181;l cells per FACS tube and protect them from light.</li>
		<li>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).</li>
		<li>Meanwhile, start the FACS (the lasers need time to warm up).</li>
		<li>Set the flow cytometry gating parameters for the experiment (see Figure 3):
		<ol>
			<li>Load around 100,000-200,000 NucleoBAS or CytoBAS transfected cells to determine the gates.</li>
			<li>First adjust the FSC and SSC voltages to plot the cells in the center of the plot.</li>
			<li>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.</li>
			<li>Set the correct gates (Gate P1 up to Gate P4), see Figure 4.
			<ol>
				<li>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.</li>
				<li>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.</li>
				<li>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.</li>
				<li>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.</li>
			</ol>
			</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Aoki, K., Kamioka, Y., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+imaging+of+cell+signaling+from+in+vitro+to+in+vivo:+basis+of+biosensor+construction,+live+imaging,+and+image+processing.">Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: basis of biosensor construction, live imaging, and image processing.</a> Dev. Growth Differ. 55 (4), 515-522 (2013).</li><li>Jares-Erijman, E. A., Jovin, T. 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. R., 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=Farnesoid+X+receptor+and+bile+salts+are+involved+in+transcriptional+regulation+of+the+gene+encoding+the+human+bile+salt+export+pump.">Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump.</a> J. Hepatol. 35 (3), 589-596 (2002).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>CytoBAS&amp;nbsp;</td><td>Addgene</td><td>62860</td><td></td></tr><tr><td>NucleoBAS&amp;nbsp;</td><td>Addgene</td><td>62861</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagles media (DMEM)</td><td>Lonza</td><td>BE12-614F</td><td>High glucose without L-glutamine</td></tr><tr><td>Penicillin-Streptomycin (pen/strep)</td><td>Lonza</td><td>17-602E</td><td></td></tr><tr><td>L-glutamine (200mM)</td><td>Lonza</td><td>17-605E</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>102-70</td><td></td></tr><tr><td>Trypsin-EDTA (10x)</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>T-25 cell culture flask</td><td>VWR international</td><td>392-0253</td><td>Laminin coated</td></tr><tr><td>T-175 cell culture flask</td><td>VWR international</td><td>392-0238</td><td>Laminin coated</td></tr><tr><td>6-well plate</td><td>VWR international</td><td>734-0229</td><td>Poly-L-lysine and Laminin coated</td></tr><tr><td>10 cm dish</td><td>VWR international</td><td>392-0243</td><td>Laminin coated</td></tr><tr><td>Diethylaminoethyl (DEAE) - Dextran</td><td>Sigma-Aldrich</td><td>D9885</td><td></td></tr><tr><td>Polyethylenimine (PEI)&amp;nbsp;</td><td>Brunschwig</td><td>23966-2</td><td></td></tr><tr><td>G418 (geneticin) 50 mg/ml</td><td>Invitrogen</td><td>10131-027</td><td></td></tr><tr><td>Hygromycin B, 50 mg/ml</td><td>Invitrogen</td><td>10687-010</td><td></td></tr><tr><td>Cloning cylinder (6 x 8 mm)</td><td>Bellco</td><td>2090-00608</td><td></td></tr><tr><td>L-15 Leibovitz culture medium</td><td>Invitrogen</td><td>21083-027</td><td>No phenol red</td></tr><tr><td>Polystyrene round bottom tube (5 ml) Facs tube</td><td>Falcon BD</td><td>352008</td><td>No cap, non-sterile</td></tr><tr><td>Falcon 2,063 tubes (5 ml)</td><td>Falcon BD</td><td>352063</td><td>Snap cap, sterile</td></tr><tr><td>Nunc Lab-Tek 8 well coverglass</td><td>Thermo scientific</td><td>155409</td><td>Sterile</td></tr><tr><td>Charcoal-filtered FBS</td><td>Life technologies</td><td>12676011</td><td></td></tr><tr><td>GW4064</td><td>Sigma-Aldrich</td><td>G5172</td><td></td></tr><tr><td>TCDCA</td><td>Sigma-Aldrich</td><td>T6260</td><td></td></tr><tr><td>CDCA</td><td>Sigma-Aldrich</td><td>C9377</td><td></td></tr><tr><td>Other chemicals</td><td>Sigma-Aldrich</td><td>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. ...

real-time-monitoring-intracellular-bile-acid-dynamics-using

Research

JoVE Journal

Bioengineering

9.8K Views.  Academic Medical Center. 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. 

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

CRAF-14-3-3 Interaction Analysis in Live Cells Using a BRET-Based Approach

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

Biochemistry

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Biology

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Medicine

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video