Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.
The overall goal of this procedure is to quantify vesicular trafficking events in the endocytic pathway of living cells. This method can help answer key questions in the vesicular trafficking field such as how mutation or changes in protein expression affect rates and efficiency of trafficking events. The main advantage of this technique is that it allows direct quantification of endocytic trafficking and living cells by measuring the intensity of fluorescently-tagged cargo proteins.
After preparing solutions and media according to the text protocol, inoculate two to three colonies of yeast cells in five milliliters of YNB medium, lacking additional nutrients for maintaining plasmid selection. Grow the cultures at 30 degrees Celsius with shaking for 16 to 24 hours. Approximately three hours before imaging, measure the OD 600 of each overnight culture.
Then prepare five milliliter dilutions at an OD 600 of 0.35 to 0.4 in YNB medium, lacking additional nutrients for plasmid maintenance and grow at 30 degrees Celsius with shaking for three hours. During the incubation, pre-equilibrate the microscopes environmental chamber or heated stage to 30 degrees Celsius. After preparing a can treated eight well glass pot and chamber slide for imaging according to the text protocol, add 200 microliters of YNB medium lacking additional nutrients for plasma selection to each well.
When the cultures are ready, transfer one milliliter to a microfridge tube and pellet the cells at 8, 000 RPM for two minutes. Then remove 975 microliters of the supernatant. Resuspend the cell pellet in the remaining supernatant.
Then drop 2.5 microliters of the cell suspension into the center of each well of the eight-well plate. Pipette up and down to disperse the cells and allow the cells to settle for five to 10 minutes. Place the chamber slide on an inverted fluorescence microscope equilibrated to 30 degrees Celsius.
Then, use bright field illumination to locate a field of cells. Add 50 microliters of pre-warmed YNB medium containing 100 micrograms per milliliter of methionine to the 200 microliters of medium in the well to obtain a final methionine concentration of 20 micrograms per milliliter. Allow the cells to equilibrate for two minutes before beginning imaging.
Then immediately before imaging, adjust the microscope to the desired focal plane. Acquire GFP in bright field or DIC images at 5-minute intervals for 45 to 60 minutes. Using identical acquisition parameters for all images within a time series.
Refer to the text protocol for additional details. Quantify the fluorescence intensity of individual cells at each time point and express values as a percentage of initial intensity of the first image. After exporting images according to the text protocol in image j, open the files in the file menu using the Open option.
Use the fluorescence image for quantification and the bright field or DIC image as a reference to identify the location of cells. Exclude dead cells of present which appear darker than live cells when viewed by DIC and autofluoresce under the GFP channel. For images with an uneven background in the Process menu, choose the background subtraction algorithm and choose the default setting.
Once images have been acquired it is important to perform background subtraction before proceeding with the quantification procedure. This step is particularly important because background signal could otherwise mask or dampen differences between samples. Using the freehand selection tool, outline a cell making sure to include the entire cell but as little area outside of the cell as possible.
Then under the Analyze menu, use the set measurements function to measure the area, integrated density and mean gray value. Open the ROI Manager by clicking on Analyze, then Tools. In the ROI Manager window, click the Add button to enter the highlighted region as a new ROI.
Repeat the outlining and measurement taking for the remaining cells in the image, excluding any dead cells or cells that touch the edge of the image. Save the selected ROIs as zip file by clicking the measure button which will display the measured values in a new window. Then click the more button and export all measurements to a spreadsheet or statistical analysis software.
Inoculate two to three colonies of yeast cells and 0.5 milliliters of YNB medium, lacking additional nutrients for maintaining plasmid selection. Grow cells on a roller drum at 30 degrees Celsius for 16 to 24 hours. Add one milliliter of YNB medium lacking additional nutrients for plasmid maintenance and grow for an additional three hours.
Next, transfer one milliliter of cells into each of two five-milliliter round bottom tubes. Then add 0.25 milliliters of YNB Met medium to the first tube and 0.25 milliliters of YNB medium containing 100 micrograms per milliliter of methionine to the second tube for a final concentration of 20 micrograms per milliliter. For a large number of samples, stagger the addition of methionine to accommodate time for facts analysis.
Our flow cytometer can measure up to 40 samples per run. We add methionine in batches. Eight samples every five minutes.
This ensures that all samples are measured between 45 and 50 minutes after methionine addition. Incubate the cells for 45 minutes. Then analyze the cells by flow cytometry, selecting forward scatter or FS as a measure of cell size and map one flowing fluorescence intensity from a FITC filter as a measure of cargo internalization.
During the 45 minute incubation, use the slider buttons to adjust the voltage of the FS and FITC channels to optimize detection for the size range and fluorescence intensity of the yeast cells being used. Measure FS and fluorescence intensity for 10, 000 cells from each condition. Using the high flow rate setting, generate a scatterplot of FS against fluorescence intensity by clicking add graph.
Then select FITC for the x-axis and FS for the y-axis and graph 1000 points. Using the gate function and the wild type Met condition, assign a vertical gate so that approximately 5%of the brightest cells fall to the right of the gate. Use this gating for all other conditions in the experiment.
As shown here, time-lapse imaging of cells was performed at five minute intervals in the absence or presence of methionine to monitor GFP or fluorine tagged Mup1 internalization and changes in fluorescence. As expected GFP and fluorine tagged Mup1 remained at the plasma membrane in the absence of methionine. In contrast, cells imaged in the presence of methionine, showed progressive depletion of the fluorescent signal from the cell surface consistent with cargo internalization.
However, the kinetics of fluorescence lost in the presence of methionine were delayed in the Mup1 GFP cells compared to the pHlourin Mup1 cells. These scatter plots compare the fluorescence intensity of wild-type or four delta ENTH1 cells in the presence or absence of methionine after applying a vertical gate. 4%of the wild-type Met cells were contained on the right side of the gate corresponding to the brightest cells in the population, while 65%of untreated wild-type cells were contained in the gate.
In contrast, the four delta ENTH1 cells showed no difference in the distribution of bright versus dim cells, consistent with a defect in endocytosis. Once mastered, this technique can be done in three to four hours if it is performed properly. While attempting this procedure it is important to remember to stagger starting times from performing simultaneous experiments, so that the endpoint data can be collected after the same treatment time for all samples.
Following this procedure other methods like floral genetic screens can be performed in order to answer additional questions like what novel factors contribute to vesicular trafficking. After its development, this technique paved the way for researchers in the field of vesicular trafficking to explore aspects of endosome attrition as well as a newly discovered clathrin independent and acidic pathway in budding yeast. After watching this video you should have a good understanding of how to use florent-type cargoes to quantify endocytic events in living cells.
