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.
The overall goal of this fluorescence imaging method is to monitor changes in membrane protein trafficking and intracellular distribution. This method provides insight into changes in protein trafficking, such as the effect of genetic mutations or pharmacological agents on the rate of vesicle delivery and protein expression on the cell surface. The main advantage of this technique is that measurements can be performed rapidly, at high resolution, in real time, without destroying the sample.
48 hours prior to imaging, mouse neuroblastoma 2A cells are transfected with a plasmid construct containing SEP, a pH sensitive fluorophor incorporated into the extracellular region of the protein of interest. When the transfected cells are ready for imaging, with total internal reflection fluorescence or TIRF microscopy, add two milliliters of pH 7.4 extracellular solution or ECS to the cells. Place a dish of cells on the translation stage and use stage mounts to secure the dish in place.
In epifluorescence mode, focus the microscope and locate the fluorescent transfected cells. Cells will remain focused across several focus planes. Locate single, isolated cells to proceed with TIRF.
In the imaging program, set the exposure time to 200 milliseconds and optimize the fluorescence intensity by setting the EM gain. Transition the laser beam into TIRF by using the Stepper motor to translate the beam across the objective in a stepwise manner. As the critical angle approaches, the beam will visibly translate across the edge of the dish until it converges at the point of total internal reflection on the sample plane, at which point the beam is no longer visible along the edge of the dish.
Verify that cells are in TIRF mode by adjusting the focus knob. In TIRF, only one plane of cells can be focused, producing a highly defined image with high resolution of the plasma membrane. To begin this procedure, locate healthy transfected single cells in the imaging plane.
Acquire a focused image of cells at pH 7.4. SEP labeled receptors on the plasma membrane and endoplasmic reticulum should be visible. Quickly block the laser beam from reaching the sample to prevent photo bleaching.
Use microscope imaging software capable of recording multiple XY locations to record the stage positions corresponding to each cell. In this way, capture images of 20 to 30 cells per dish while using the software to record the position of each cell. After all cell images at ph 7.4 are collected, manually remove the pH 7.4 ECS by pipetting without touching the dish.
Carefully add two milliliters of pH 5.4 ECS to the dish and wait for 10 minutes. During this time, save previously acquired images. Under the identical set of conditions used to collect images at pH 7.4, move the stage to each saved position and acquire an image of the same cell at pH 5.4.
The cells should look less defined since all detected fluorescence is originating from endoplasmic reticulum confined SEP labeled receptors. Save the pH 5.4 cell images. To image single vesicle insertions, first replace the growth media of transfected cells with two milliliters of pH 7.4 ECS.
Next, place the dish of transfected cells on the stage of the microscope and use stage mounts to secure the dish in place. Focus a single cell in TIRF as demonstrated earlier. If equipped, set the auto focus so the focus does not drift over the period of imaging.
Record a series of 1, 000 frames continuously capturing images at a frame rate of 200 milliseconds. Bursts of fluorescence will be visible during this time, corresponding to low pH trafficking vesicles fusing with the plasma membrane, exposing the SEP to the extracellular pH 7.4. To begin the analysis of SEP fluorescence to determine subcellular localization, open the cell images using an image analysis software such as ImageJ.
Subtract the background from both pH 5.4 and pH 7.4 images using the rolling ball setting. Using intensity based threshold to quantify fluorescence from a single cell at pH 7.4, first select image, adjust, and threshold. Then manually select a region of interest around the cell.
Using the built in measure function under the analyze tab, measure the cell area, mean intensity, and integrated density. Repeat these measurements for the same cell at pH 5.4. After the integrated density is obtained for cell images at both pH 7.4 and 5.4, calculate the plasma membrane integrated density by subtracting the pH 5.4 value from the pH 7.4 value.
The difference corresponds to the relative number of receptors on the plasma membrane within the TIRF excitation region. As a measure of trafficking, calculate the relative percentage of SEP labeled receptors located on the plasma membrane compared to the remaining receptors visible in the TIRF excitation volume by dividing the plasma membrane integrated density by the total integrated density at pH 7.4, multiplied by 100. To analyze single vesicle insertion events, open the series of 1, 000 TIFF images using image analysis software.
Subtract the background from all frames using the rolling ball setting. Adjust the color balance of the recording to maximize intense regions corresponding to vesicle insertion events. Manually count the bursts of fluorescence lasting longer than three frames or 600 milliseconds.
This example of a cell image at pH 7.4 shows receptors located on the plasma membrane and endoplasmic reticulum. At pH 5.4, receptors on the plasma membrane do not fluoresce, so only the endoplasmic reticulum resident receptors are visible. The difference between integrated density of the fluorescence at pH 7.4 and pH 5.4 corresponds to receptors localized to the plasma membrane.
Single vesicle insertion events are visualized at pH 7.4 as a burst of fluorescence at the plasma membrane, lasting at least 600 milliseconds. Immediately preceding an insertion, no discernible burst is present. An increase in fluorescence intensity is seen as a transport vesicle carrying SEP arrives.
The SEP labeled receptors then diffuse across the plasma membrane until indistinguishable from previously inserted receptors. Once mastered, this technique can be done in 45 minutes per dish, if performed properly. After watching this video, you should have a good understanding of how to monitor changes in protein distribution between the peripheral ER and plasma membrane.
