The overall goal of this procedure is to measure the amount of each phosphoinositide species in cells under various genetic and environmental conditions in order to understand phosphoinositide function and regulation. Phosphoinositide lipids control cell functions like migration, replication, and memory trafficking. This method identifies genetic and environmental conditions that impact phosphoinositides and increases our knowledge about their regulation and function.
The main advantage of this technique is that it permits accurate, concurrent measurement of all phosphoinositides inside cells. Though this method can provide insight into the regulation of phosphoinositides in yeasts, it can also be applied to cultured mammalian cells. Individuals new to this method will struggle because of the many steps required for labeling, processing, and analysis.
We recommend practicing without the radioactive label first. To begin, grow a 20 milliliter liquid culture of the yeast strain SEY6210, in complete synthetic medium at 30 degrees Celsius with constant shaking to mid-log phase. Next, transfer a total of 10 to 14 OD of the yeast cells to a 12 milliliter round bottom centrifuge tube and centrifuge at 800 times G for five minutes.
Decant the medium. And re-suspend the pellet in 2 milliliters of inositol-free medium. Centrifuge the cells again.
And then re-suspend the pellet in 440 microliters of inositol-free medium. Incubate the cells at room temperature for 15 minutes. After the incubation, add 60 microliters of tritium labeled myo-inositol and grow the cells for an additional one to three hours at 30 degrees Celsius with constant shaking.
Transfer the cell suspension to a microcentrifuge tube containing 500 microliters of of 9%perchloric acid and 200 microliters of acid washed glass beads. Mix by repeatedly inverting the tube, and then incubate on ice for five minutes. Afterward, vortex the tube at maximum speed for 10 minutes.
Then transfer the lysate to a new microcentrifuge tube using a gel loading tip to avoid aspirating the glass beads. Next, centrifuge the tube and discard the supernatant. Sonicate the pellet in one milliliter of ice cold 100 millimolar EDTA until it is completely dispersed, and centrifuge again.
After centrifuging, aspirate the EDTA from the tube containing the radio-labeled pellet. Add 50 microliters of water and sonicate to re-suspend the pellet. Prepare fresh deacylation reagent according to the text protocol.
Then add 500 microliters of the deacylation reagent to the tube, and sonicate to mix. Incubate the sample at room temperature for 20 minutes. Next, transfer the sample to a heating block at 53 degrees Celsius for 50 minutes.
Afterward, place the sample in a vacuum centrifuge and allow it to dry for a minimum of three hours. Re-suspend the pellet by sonicating in 300 microliters of water, and incubate it at room temperature for 20 minutes. Afterwards, dry the sample completely in a vacuum centrifuge for a minimum of three hours.
After drying, add 450 microliters of water to the tube and re-suspend the pellet by sonicating until it is completely dispersed. Prepare fresh extraction reagent according to the text protocol. Add 300 microliters of extraction reagent and vortex at maximum speed for five minutes.
Centrifuge the tube at 18000 times G for two minutes, and carefully pipette the bottom aqueous layer into a new tube. Next, dry the final aqueous fraction by vacuum centrifugation for a minimum of three hours. Afterward, fully disperse the pellet by sonicating in 50 microliters of water, and store the sample at 20 degrees Celsius.
Finally, determine the radioactivity of the sample by adding two microliters to four milliliters of scintillation fluid in a 6 milliliter polyethylene scintillation vial. Record the CPM of the vial using a liquid scintillation counter with an open window. To set up the HPLC system for separation of the tritium labeled glycero-inositides, first prepare buffers A and B, and chromatography column according to the text protocol.
Next, load 10 million CPM of the sample into a two milliliter injection vial, fitted with a spring-loaded 250 microliter insert, and add water for a total volume of 55 microliters. Cap the vial with a screw cap outfitted with a PTFE silicone septum and place it into the auto-sampling tray of the HPLC system, along with a water blank in a similar vial. Using the software on the HPLC system, program an elution protocol.
One percent buffer B for five minutes, one to 20 percent B for forty minutes, 20 to 100 percent B for ten minutes, 100%B for five minutes, 100 to one percent B for twenty minutes, and then one percent B for ten minutes. Set the pump flow rate at 1.0 milliliters per minute over 90 minutes, and the pressure limit at 400 bar. Follow this by setting up a detection protocol on the flow scintillator to run for 60 minutes, with the scintillation fluid flow rate of 2.5 per minute, and a dwell time of 8.57 seconds.
Next, program an automated injection sequence on the HPLC to begin with the water blank running the equilibration protocol, followed by the radio-labeled sample on the elution protocol. Before the equilibration protocol is completed, create a batch sequence on the flow scintillator to measure all the samples using the detection protocol, which is triggered by the elution protocol. To analyze the HPLC data, first open the raw data file for each sample, using the chromatograph quantitation software.
In the chromatograms tab, zoom in to stretch the lesser peaks while maintaining the time resolution. Use the add ROI tool to highlight each peak for analysis, and then identify the peaks based on their elution time. Use the regions table tab to locate and record the area of each peak.
At the same time, note the start and end time of each peak. Next, perform a background subtraction for each peak by highlighting a region adjacent to the peak, spanning the same amount of time. Using the spreadsheet software, subtract the number of counts for that region from the counts of the corresponding peak.
Normalize the area of each peak against the parental glycerated inositol peak, and record as percent of the parental phosphatidylinositol. Then normalize each of the peaks in each experimental condition against the control condition, and express as the end fold increase compared to the control. Export the files by clicking on File, and Save As, and choosing the CSV format.
Finally, open the CSV file in a spreadsheet program to plot the data. The analysis of radio-labeled yeast phosphoinositides was performed by HPLC. Since yeast cells generate only four phosphoinositides, resolution can be achieved with a one hour double gradient elution method.
The glycerated inositol peak alluding at eight to nine minutes overshadowed the signal from all the other phosphorylated species. Therefore, it is necessary to zoom in on the portion of the trace containing the other peaks before integrating the radioactive signals. Wild-type, ATG18 deleted, and VAC14 deleted yeast strains were assayed for changes in phosphatidylinositol 3, 5-bisphosphate using this HPLC method.
The ATG18 deleted mutant was shown to produce the most phosphatidylinositol 3, 5-bisphosphate and the VAC14 deleted mutant produced the least. Once mastered, this technique can be done in four days if it is done properly. While attempting this procedure, it is important to remember to optimize growth and labeling conditions based on the specific yeast strain or cell types.
Following this procedure, other methods, like GFP-fused biosensors, can be used to complement HPLC flow scintillation for detection of phosphoinositides. After watching this video, you should have a good understanding of how to radio label, extract and measure phosphoinositides by HPLC coupled flow scintillation. Don't forget that working with radioactivity and organic solvents can be extremely hazardous, and precautions such as wearing personal protective equipment and proper training should always be taken while performing this procedure.
