The overall goal of this procedure is to use a Genetically Encoded FRET Bile Acid Sensor to easily visualize bile acid transport and dynamics with high spacial and temporal resolution. This sensor can answer key metabolic questions, such as the relation of biles and dynamics to glucose, lipids, and energy metabolism. The main advantage of this technique is that bile acid transport can now be directly monitored to single living cells without the need for labeling bile acids.
This method can provide insight into cell and bile acid transport and dynamics. It can also be used to visuialize effects or activation and single living cells, or to combine imaging of protein localization and bile acid transport. The genetically encoded bile acid sensor, henceforth, the sensor, consists of a donor cerulean fluorophore, and excepter citrine fluorophore.
The two fluorophores are linked by the ligand binding domain of the FXR nuclear bile acid receptor, fused to a FXR co-factor, peptide. Two engineered amino acid changes in the two fluorophores promote inter-molecular interactions between the cerulean and the citrine. Two different constructs carrying the sensor are utilized.
In one construct, the sensor contains the c-terminal nuclear localization signal, and, therefore, accumulates in the nucleus. In the other construct, the sensor lacks any targeting sequence, and thus localizes in the cytoplasm. The following video protocol focuses on how to image the sensor in living cells.
The text protocol covers how to engineer cells that express the sensor and how to further analyze them by facts. In preparation for imaging the cells, plate the cells that express the sensor at the desired density in a sterile, eight well cover slip bottom chamber slide in complete culture media. When the cells have grown to 60 t0 70 percent confluency proceed with the experiment.
Wash the cells that are adhered to the chamber slide once with 200 microliters of 1x PBS or Leibovitz L-15 Culture Medium. Then, aspirate the solution and replace it with 300 microliters of Leibovitz L-15 Culture Medium so that no CO2 control is necessary. If DMEM with Phenol Red is used, instead of Leibovitz L-15, then plan to keep track of the PH during the data collection, because the sensor is sensitive to PH.Next, prepare dilutions of the compounds to be used in the experiment at 3x to 5x concentrates, including GW4064 and TCDCA.
Prepare to add aliquots of 50 to 100 microliters during the imaging, so that the solutions are quickly mixed in the slide chamber. Now, start the imaging software of a confocal microscope, and turn on the laser. The microscope must be equipped with a 63x oil objective, a 37 degree Celsius incubation chamber, and a violet 405 nanometer laser.
Next, adjust the settings of the microscope. Set the acquisition mode to XYT for time-lapse imaging of a single, focal plane. Set the image acquisition rate to 20 second intervals, so compounds can be added during the breaks.
Also, be sure to set spectral range for a mission detection. Focus on the cells using transillumination. Then, in the software control, draw circles to define the regions of interest.
Select cells with similar florescence intensities and normal size and shape. Do not draw to close to the cell's perimeters. Then, start taking measurements.
Take note of their value, and continue taking measurements until the cerulean and citrine florescence has stabilized. Once florescence stability has been achieved, add 50 to 100 microliters of bile acids or other compounds at chosen time points during measuring. Avoid the edge of the chamber when pipetting, and add the liquid slowly.
Correct execution of this step is critical to the experiment. Again, let the florescence stabilize before proceeding. End the experiment by carefully adding 100 microliters of GW4064 for a concentration of five to 10 micromolar.
Continue recording until the florescence has stabilized once again. Then, save the experiment and export the data as a avi file. Open the saved avi files in image J by selecting plugins, stacks, stack interleaver.
Next, click on edit, selection, and add to manager to open the RLI manager window. Then, toggle the option, show all. With the oval selection tool, draw a few ROIs that cover specific cells.
Don't draw close to the cells perimeters because the cells will likely undergo some focal drift or migration. Also, draw one circle in an area outside of the cells to determine the background signal. An area inside of a cell that does not express the sensor can also be used.
Now, select one ROI, and click on plugins, and then ratio profiler. Three screens will pop up. RAW, Ratio, and Ratio Profile.
The RAW window shows the increase in intensity of citrine as a blue line, and a decrease in cerulean as a red line, if there is FRET. The ratio window shows information about the citrine to cerulean ratio, which will increase with an increase in FRET. The ratio profile window shows values for the florescence intensity measurements in both channels.
If microscope specific setup files are used, the channel order may appear reversed. Now, copy the data from the ratio profile window to a spreadsheet for statistical analysis. Then, proceed to do the same for all the other selected ROIs as well as the background ROI.
A live cell quantitative intensity based FRET experiment was performed on U2OS cells expressing the sensor, and lacking endogenous bile acid transporters. The cells were treated with TCDCA. This did not result in any changes in FRET.
When the cells were treated with GW4064, the average citrine to cerulean ratio increased remarkably. To demonstrate that the sensor can be combined with other proteins, U2OS cells co-expressing the sensor with NTCP and OST alpha-beta were engineered. They showed a clear increase in the citrine/cerulean ratio upon addition to TCDCA and GW4064.
After watching this video, you should have a good understanding of how to analyze bile acid dynamics using the bile acid sensor. Once mastered, this technique can be done in one hour. This procedure can easily be extended to include more imaging information, such as the subcellular localization of your protein of interest.
As long as the wavelength of this protein is different from the sensor. To prevent photobleaching, it's important to keep the sensor expressing cells in the dark as much as possible.
