We study interactions between receptor tyrosine kinases and the mammalian plasma membrane. We want to know how lipid rafts and lipid asymmetry drive receptor localization, activation, and signaling. It's challenging to study lipid rafts directly because they're small, dynamic, and sensitive to temperature conditions.
Researchers often have to use model systems with large order domains to study interactions. Synthetic and plasma membrane vesicles are imperfect representations of cell membranes lacking lipid asymmetry and organelles. Working with live cells, we achieve an accurate representation of the plasma membrane.
They highlight the initiation of receptor signaling in the context of live cells. This gives insight to the effect of membrane environments on receptors and their downstream signaling pathways. To begin, aliquot the lipid stock into a borosilicate tube using a positive displacement pipette with a glass tip.
Place the borosilicate tube on a heating block set to approximately 50 degrees Celsius and apply a stream of nitrogen gas to the tube until all apparent chloroform evaporates. Transfer the tube to a vacuum chamber and expose it to a high vacuum for one hour to remove any remaining solvent. Then add serum free Ham's F-12 media to the dried lipid film to reach a final concentration of 20 millimolar.
Cover the tube with a lid or Teflon tape and heat it in a 70 degree Celsius water bath for five minutes. Next, vortex the solution to suspend lipids and form multilamellar vesicles or MLVs. After the media turns cloudy, transfer the entire volume to a micro centrifuge tube and store it at four degrees Celsius for up to three days.
Mix the prepared MLVs with methyl alpha cyclodextrin to a final concentration of 40 millimolar. Incubate the lipid mixture for 30 minutes in a 37 degree Celsius or 55 degree Celsius water bath. Observe the media transitioning from cloudy to clear.
Then, let the lipid exchange media cool to room temperature for 30 to 60 minutes. Obtain Chinese hamster ovary insulin receptor cells grown at 37 degrees Celsius and 5%carbon dioxide in supplemented DMEM containing 4.5 grams per liter of glucose. Seed 1.5 times 10 to the power of six cells in 60 millimeter plates and incubate them to obtain 80 to 90%confluency.
Then, wash the cells three times with one milliliter of PBS. Starve the cells overnight in two milliliters of serum free Ham's F-12 media. After washing the cells thrice with PBS, add one milliliter of the prepared exchange media.
Incubate the cells for one hour at room temperature, swirling every 15 minutes to ensure even exposure to the exchange media. Then, wash the cells three times with one milliliter of PBS. Remove the PBS from the cell culture plate completely and set the plate at a 45 degree angle for 10 minutes or until fully dry.
After removing the residual buffer, add one milliliter of a hexane isopropanol solution to the dried cells and incubate for 10 minutes on a shaker at room temperature. Now transfer the solution to a borosilicate tube. After covering the tube, store it at minus 20 degrees Celsius.
Next, dissolve the remaining cell debris using 500 microliters of one normal sodium hydroxide. Shake the container for 10 minutes at room temperature to ensure complete dissolution. To check the exchange efficiency, pour 100 milliliters of a chloroform methanol 30%ammonium hydroxide solution into a glass thin layer chromatography tank.
Cover the tank tightly and let the vapor equilibrate for at least one hour. Dry the lipid extract sample on a heating block at a low setting under a stream of nitrogen gas until the organic solvent evaporates. Then, dissolve the lipid film in 50 microliters of a one-to-one chloroform methanol solution.
Using a 10 microliter Hamilton syringe, load one to 10 microliters of the sample onto a silica high performance TLC plate. Apply the sample in one centimeter bands loading a maximum of 10 bands per 20 centimeter plate. Place the TLC plate upright in the TLC tank and allow the solvent front to travel eight centimeters to separate phospholipid species.
Then, let the plate dry for 10 minutes and spray it with an aqueous solution of 3%cupric acetate and 8%phosphoric acid. Let the plate dry again for 30 minutes at room temperature or with a heat gun. The plate should turn from translucent blue to opaque white.
Next, char the plate in an oven tempered at 180 to 200 degrees Celsius for 5 to 10 minutes, or until black lipid bands become visible. Incubate the treated cells with 500 microliters of 100 nanomolar insulin in serum free media for five minutes at room temperature. After washing the cells with ice cold PBS, place them on ice to halt stimulation.
Then add one milliliter of ice cold PBS and harvest the cells using a cell scraper. Pellet the cells at 3000 G for five minutes at four degrees Celsius. Next, add 100 to 200 microliters of complete RIPA lysis buffer to the cell pellet and resuspend 30 to 40 times to lyse the cells.
After incubating the lysate on ice for 10 minutes, centrifuge it at 16, 000 G for 10 minutes at four degrees Celsius to separate the debris. Collect the supernatant in a fresh tube. After determining the protein concentration, mix the lysate with five times laemmli buffer and heat it at 95 degrees Celsius for five minutes.
Load equal amounts of protein per sample onto a sodium dodecyl sulfate polyacrylamide gel for electrophoresis. Run the gel at 100 to 150 volts in a running buffer until proteins are well resolved between 100 to 250 kilodaltons. Then, transfer the resolved proteins onto a polyvinylidene fluoride membrane using a transfer buffer and stain the membrane with the appropriate antibodies.
Lipid exchange in the Chinese hamster ovary IR cells resulted in increased sphingomyelin band intensity and decreased phosphatidylcholine band intensity when brain sphingomyelin was exchanged. Conversely, when 1, 2-dioleoyl-sn-glycero-3-phosphocholine or DOPC was exchanged, phosphatidylcholine band intensity increased, while sphingomyelin band intensity decreased. The cells exchanged with DOPC showed a decrease in insulin dependent IR autophosphorylation.
In contrast, brain sphingomyelin exchanged cells maintained or moderately increased IR phosphorylation. Normalized phosphorylation levels were determined by dividing the pYpY IR intensity by the IR beta intensity from western blot data.
