The overall goal of this experiment is to obtain quantitative spatial and temporal evidence of Angiotensin type one receptor lysosome colocalization after Angiotensin two treatment. This method was devised to detect differences in the subcellular processing of Y type versus mutant receptors, As during the internalization process, that utters in response to ligand simulation. To advantages of this technique are that life cell imaging, eliminates artifacts from fixed and detergent permeabilized cells and that dynamic changes in receptor localization can be assessed in real time.
Begin by selecting an appropriate 1.4 numerical aperture objective and centering the objective over the microscope stage. Add a drop of hight viscosity, low auto florescence immersion oil onto the objective and transfer the chamber with the tranfected cells to the microscope stage. Gently raise the objective until the oil just touches the bottom of the first slide and locate the dim cells.
With a healthy cell of interest, in the center of the field of view, switch to confocal mode and confirm that the cell is well spread, with its ventral side juxtaposed closely with the cover slip. Open the Z Stack dialog box, click Live, focus on the top of the cell, and then click Begin. Then focus to the bottom of the cell, enter Z step size 1.00 micrometer, and then click End.
Then select XYZT Mode under Acquisition settings at the top. Lastly, set the time interval for the scan and the length of time for scanning to observe the targeting of GFP tagged Angiotensin Type 1A Receptor to the lysosomes. Then to begin the ligand stimulation, add three microliters of a 100 X stock solution of ligand to the well that is being imaged and click Start to image the response in 3D for the desired period and frequency.
To analyze the live cell images, open the appropriate 3D image quantification software. In this protocol, Velocity. Right click within the image field and select Properties.
Enter the appropriate XY and Z image pixel properties for creating a measurement sequence to analyze the GFP tagged receptor vesicle, and hole cell intensities in response to ligand treatment. Under the 2D menu, select the extended focus mode to observe the cell. Next, use the freestyle region tool, to draw a region of interest around the cell, and right click the image to select Crop to Selection.
A new image item will be generated. Click on the image name to select the cropped cell image and open the measurement tab. To measure the GFP expression of the internalized vesicles, drag Find Objects from Finding into the sequence box above.
Set the find objects channel to GFP and open the settings icon to set the threshold using to standard deviation and the lower limit to six, to select objects with the brightest GFP intensities. Set the minimum object size to zero micrometers cubed. Then, click measure, and select the channels and the types of measurements.
To define the thresholding and filters for identifying the whole cell and to measure the total GFP intensity, drag a second Find Objects box. Next, set the Find Objects channel to GFP and set the standard deviation to zero, to select objects with any GFP intensity and minimum object size to two micrometers cubed. Drag Filter Population to the Population two Find Objects box and select a volume of micrometers cubed, greater than 100 to select the whole cell and eliminate all smaller objects.
Then, select measurements and Make Measurement item, to make measurement at all time points. To analyze the measured GFP expression in vesicles, and the whole cell over time, click on the measurement item file name below the image name. To extract the data for GFP and vesicles from the total data, open the raw tab and select population one.
Then, open the analyzed tab and right clicking on the data, select analysis, followed by restricting analysis to select population one. To measure the amount of GFP in the whole cell, change both to population two. Under Analyze These Data and Summarized By, select sum GFP color channel, and sum.
Then, under Organize the data by, and row, select time point. To extract the data for the amount of GFP in lysosomes, in the Find Objects for population one, set the find objects channel to the red channel. In the wheel icon within the dialog box, set the threshold using to standard deviation, the lower limit to six, to select red objects with the brightest intensities, and the minimum object size to 0.017 micrometers cubed.
Select measurements, and make make measurement item, to make measurement at all time points. To analyze the measured GFP expression in lysosomes, click on the measurement item file name below the image name. Select population one under the raw tab, and then select population one and sum GFP color channel and sum under the analyze tab, right clicking as before to measure the amount of GFP in the red vesicles.
Before ligand stimulation, GFP tagged Angiotensin Type 1A Receptors are localized predominantly in the plasma membrane. But within 2.5 minutes of the addition of Angiotensin two, these receptors become internalized, forming bright vesicles and later clustering and large paranuclearzome vesicles. Angiotensin two induced ruffling complicates the quantification of the total vesicle GFP intensity as observed for these representative GFP expression measurement data.
Size filtering does not discriminate between the larger ruffles and clustered vesicles, but does reveal that GFP in smaller objects increases sooner than GFP in larger objects. In this representative time course of GFP expression without autofocus, the internalized GFP was measured as the percent total cellular GFP. Analysis of the total GFP within the cell revealed that this cell did not photo bleach appreciably over time.
Quantification of the GFP in the lysosomes of transfected cells also indicates that the intensity of the Angiotensin Type 1A Receptor present within the lysosomes, does not vary for up to 24 minutes after Angiotensin two administration. During this procedure, it's important to remember to monitor the health of the cells prior to and during the imaging, since the procedure is time consuming, the experiment should be aborted if the cell cultures are abnormal in any way. Following this procedure, immunostaining and FRET or FLIM can be used to detect and confirm the subcellular localization of the receptors.
Western blots can be performed to address the identity and changes in signaling molecules involved during the receptor trafficking. After watching this video, you should have a good understanding of how to perform live cell imaging and analysis to measure the ligand induced transmembrane receptor targeting to lysosomes as well as to other subcellular compartments.
