Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

The overall goal of the following experiment is to observe real time changes in glyco composition in living cells. This is achieved by expressing a fluorescent protein fused to a Pero Somal targeting sequence PTs two. The fusion protein is imported into mature glyco zones, which become fluorescent organelle.

Degradation, and recycling leads to a loss of fluorescence that can be monitored via flow Cytometry cells are exposed to different environmental conditions in order to identify factors that trigger glyco remodeling. The results show that glyco composition varies in response to environmental changes based on changes in cellular fluorescence upon passage of cells from nutrient poor to nutrient rich media. So the main advantage of this technique over existing methods like electron and fluorescence microscopy, is that it allows for real time analysis of thousands of cells.

Demonstrating the procedure will be Sarah Bauer, a graduate student, and Megan Conlan, an undergraduate student from my laboratory. The insect procyclical form or PCF parasites for this procedure are cultured in a 25 centimeter squared cell culture flask with 10 milliliters of appropriate medium at 27 degrees Celsius and 5%CO2. Preparation of the plasma DNA will not be shown in this video, but the protocol is available in the accompanying manuscript.

To begin this procedure, add 10 microliters of sterile purified linearized DNA to 400 microliters of filter sterilized cyto mix count cells using a flow cytometer and harvest 50 to a hundred million cells by centrifugation at 800 Gs for 15 minutes. At room temperature, pour off the supernatant using a one milliliter serological pipette resuspend the cell pellet in 450 microliters of DNA and Cyt mix. Transfer the solution containing cells DNA and cyto mix to a sterile four milliliter gap.

Qve and place the qve in the electroporation chamber. Select exponential decay and manually enter the following settings. Voltage 1.5 kilovolts capacitance 25 milli resistance, infinite omega and vet four millimeters.

Press pulse once the pulse is complete. Remove the vet from the electroporation chamber and return to the biosafety cabinet. Pipette 10 milliliters of SDM 79 media into a new sterile flask.

Transfer the transformed cells to the flask with the SDM 79. Incubate the cells without drug selection for 24 hours. At 27 degrees Celsius, 5%CO2.

After 24 hours a G 4 1 8 hygromycin and blastin pass. One milliliter of transformed cells with drug into nine milliliters of SDM 79 with drug return the cells to the incubator. Subsequently, the cells are analyzed daily by flow cytometry, which will be demonstrated later in this video.

S for long-term storage are made when cells are fluorescent and have reached the cell density of 10 million cells per milliliter. First, make freezing media by adding an equal volume of 100%glycerol to the cyto mix and filter sterilizing the solution. Next, add 200 microliters of freezing media to 800 microliters of cells in a cryo vial and mix gently by inversion.

Place the cryo vial between two styrofoam racks and freeze at minus 80 degrees Celsius for about 24 hours. Once frozen, transfer the cells to liquid nitrogen, it is important to move the cells into liquid nitrogen After 24 hours as longer storage at minus 80 degrees Celsius leads to a decrease in cell viability To thaw frozen stock. Remove the frozen vial from the minus 80 degrees Celsius freezer and thaw room temperature for approximately 10 minutes.

Pipette nine milliliters of SDM 79 media with drug into a new sterile flask. Add thawed cells to this flask, passage to cells one to 10 by adding one milliliter of this culture to nine milliliters of SDM 79 in a new flask to prepare the culture for a dilution assay passage to cells to a density of 100, 000 cells per milliliter in three milliliters of SDM 79. In a 25 milliliter culture flask fluorescent is measured immediately after passage, and then at three hours and at 24 hours Only passing the cells back one time prior to beginning dilution assay is essential for observing glyco somal remodeling.

When cells are cultured beyond two passes, lysosomal remodeling becomes unpredictable. Before running any samples, the fluidics should be back flushed and rinsed as demonstrated. Here, turn on the flow cytometer and open the C Flow plus program.

Replace the tube of nano pure water in which the SIP is stored with an empty tube. Select the back flush button. Once the back flush is complete.

Remove the micro centrifuge tube from the sip and discard it. Place a new micro centrifuge tube containing one milliliter of new nano pure water on the sip. Select the new well in the C Flow program under run limits.

Set the time for two minutes. Under fluidics, select fast and run. Once a two minute time limit is complete, click the delete sample data button.

After the flow cytometer has been set up, the cells can be counted. Replace the water on the SIP with the sample to be counted. Set run limits to 30 seconds, fluidics to fast, and then select run after the 32nd time limit is complete.

C Flow plus will provide the events per microliter under last run. To measure fluorescence, set run limits to 10, 000 events and fluidics. To slow select set threshold.

Under primary threshold, select permanently eliminate events on FSCH and enter less than 30, 000. Click apply followed by close, select the histogram button in one of the new plot windows and select FSCA From the dropdown list, select FL one A, which measures fluorescence. In the 530 nanometer wavelength select run.

After all the cultures have been assessed for fluorescence, run cleaning solution through the fluidics line for two minutes and water for two minutes. To begin data analysis, select the analyze tab on C Flow plus. Click the histogram button under, make a new plot.

Select FSEA. A dropdown list will appear. Select channel FFL one a.

Highlight a well to analyze, select the gate button and manually draw a gate for the population of interest. C Flow plus will provide a cell count within this gate and percent of this plot flow cytometry analysis of PCF cells grown in glucose containing media. SDM 79 revealed two populations, one bright and one dim.

Dim cells harbor immature glyco zones, which have not imported the fluorescent reporter. While bright cells harbor more mature glyco zones, when glucose is present in the media, mis localization of glyco proteins is lethal, and gly protein expression and import must be tightly controlled. This is reflected in the absence of cells with intermediate fluorescence intensities.

In SDM 80 media Without glucose, the mis locallocalization of glyc proteins is tolerated. The bimodal population distribution is lost, and cells of intermediate fluorescence are observed. This system can be used to identify conditions that trigger glyco remodeling.

When high density cultures containing approximately 10%dim cells or passage into fresh media, there is an increase in the percentage of dim cells with immature glyco zones falling within the left gate. By 24 hours, the original population distribution is reestablished. As the immature glyco foams now contain the peroxisome proteins necessary to import the fluorescent protein.

While attempting this procedure, it's important to remember to avoid extensive culturing. After two passes, cultures should be discarded and new stable eights used. This protocol allows the identification of reagents and conditions that trigger glyco modeling.

Following this procedure, other medicines like Western analysis and fluorescence and electron microscopy can be performed. In order to answer more precisely the changes in glyco protein expression, localization and morphology.