This procedure assays the effects of small GTP ACEs on polarized membrane trafficking. First, co inject DNA plasmids encoding the small GTP ACE and the reporter proteins into cell nuclei of polarized cells. Then express the DNA at temperatures that will arrest the reporter proteins in the endoplasmic reticulum or the TGN while the small GTPA accumulates in the cytosol proceed to chase the reporter protein to the cell surface in the presence of cyclo heide to prevent further protein synthesis.
Finally, label the reporter protein at the cell surface for immunofluorescence analysis. Results obtained from this assay permit visualization of the change in surface localization through confocal microscopy. The main advantage of this technique of existing methods like trans transfect is that during the short times of overexpression achieved with micro injection, secondary effects are minimal, allowing us to study the primary effects of the proteins of interest.
This method can help answer key questions in the cell polarity field, such as how small gtpa is regulate polarized membrane trafficking. Visual demonstration of this method is critical as the individual steps necessary for a successful microinjection are difficult to explain without visual aid Isolate endotoxin free plasma DNA with the Sigma Aldrich endotoxin free maxi prep kit according to the manufacturer's protocol. For this experiment, use three clear 12 millimeter transwell filters of 0.4 micrometer pore size to culture.
The cells seat four times 10 of the fifth MDCK cells onto each filter. After two days, examine the culture under a microscope to verify that the cells are growing in a closed monolayer. For microinjection.
On the day of microinjection, prepare 360 by 15 millimeter plates of five liters MM growth media plus 15 millimeter heis and place them into a 39 degree Celsius incubator. In addition, prepare three 12 well plates of one milliliter MEM growth media plus 50 millimolar heis, and 0.1 milligram per milliliter cycloheximide and place them into a 31 degree Celsius incubator. Turn on the microinjection microscope, set up like this inverted microscope with heated stage 10 x and 32 x objectives, and an einor femto jet.
Set the heated stage to 39 degrees Celsius. Also open the nitrogen gas tank supply to the air table. Now dilute the DNA with filtered water to a final concentration of 0.2 milligrams per milliliter.
Subsequently spin DNA in an einor micro centrifuge at 13, 000 RPM for 30 minutes. Remove the top portion and place in a new tube. Next, prepare the MDCK cells by taking the first filter out of its culture dish with the surgical blade, cut out the filter from the filter holder and place it into the prepared 60 by 15 millimeter plate containing five milliliters of 39 degrees Celsius warmed MEM growth media plus 50 millimolar heis.
To weigh down the filter in the culture plate, place the surgical blade in the center of the filter and then transfer the culture plate onto the heated stage of the microscope. Load two to three microliters of the diluted DNA into a microinjection needle. Twist the protective cover of the needle and let it fall to the floor.
To position the needle into the holder, press the menu key of the inject and make sure the valve is shut down. Screw the needle into its holder. Beware not to screw in the needle too tightly.
As that may lead to breakage, press the menu key again. So the applied compensation pressure will prevent the media from being sucked into the needle during the microinjection procedure. Finally, tap the joystick to erase stored homing to lower the needle onto the cells.
Use the 10 x objective and bring the needle into the light beam above the liquid. Now focus on the cells. Focus up again by turning the focus wheel 180 degrees up and find the needle.
Slowly move the needle into focus. Then bring it out of focus again. Working towards bringing the cells into focus Again.
Bring the needle down into focus Again. Repeat until the needle touches the surface of the media at which point a halo is observed. Continue to reach the point at which the cells are in focus, but the needle is still fuzzy out of the focal plane.
Now switch to the 32 x objective and find course settings. Set the Z limit by touching the apical membrane with the tip of the needle and subtracting about 10 micrometers. As the nuclei are laying approximately 10 micrometers underneath the apical membrane.
Now bring the needle a couple of microns out of the focal plane as fast as possible. Aim with the needle above the nucleus and press and release the injection button of the joystick. Start at 95 PSI to find the right injection pressure.
If the pressure is too high, the cells blow up. If the pressure is too low, a white dot persists, but nothing else happens. Perform a successful injection involving a phase change without a cell size change.
Inject 100 to 500 cells within the hole of the surgical blade that lies on the cells. Then place the cells with the culture dish and surgical blade into a 39 degree Celsius incubator and incubate for two hours. Finally, transfer cells into the prepared 12 well plate of one milliliter, MEM plus 50 millimolar 0.1 milligram per milliliter, cyclo heide and incubate for two hours at 31 degrees Celsius.
In order to avoid bleaching of the expressed GFP signal, protect the specimens from light by covering with aluminum foil during all subsequent procedures for surface staining. Place the cells in a culture dish onto a metal plate on ice and wash. Once with ice cold PBS plus plus, position a clean piece of paraform on the metal plate on ice pipette 30 microliter drops of an antibody that recognizes the ecto domain of the protein of interest.
Now place the filter containing cells upside down onto the drop and add a few drops of antibody onto the backside of the filter. Incubate for one hour on ice. Wash the cells three times with ice cold PBS plus plus and fix with 3%paraform aldehyde for 15 minutes at room temperature.
Proceed to wash cells once with PBS plus plus and equilibrate in PBS plus plus for five minutes. Now incubate the cells in blocking permeable buffer. Incubate one hour at room temperature, dilute primary antibodies one to 200 in BPB to detect the expressed RAB GTP ACE centrifuge for 10 minutes At 13, 000 RPM pipette, 30 microliters of the antibody solution onto clean param placed in a wet chamber.
Place the cells on the filter upside down onto the antibody drops and incubate for one hour. At room temperature, place cells back right side up into a 12. Well plate and wash five times over 30 minutes with BPB at room temperature after incubation with appropriate secondary antibodies, including a wash step.
Dip cells on the filter three times into deionized water and place right side up onto micro slides at 10 microliters of mount Place an 18 by 18 millimeter micro covered glass on top and using facial tissues gently press the cover slip onto the cells. Finally seal with nail polish in the mock injection of only the plasmid encoding V-S-V-G-T-S-O 45 GFP. The protein is delivered to the basolateral surface of well polarized MDCK cells as judged by the red surface staining in cells that are not well polarized.
Some of the GFP fusion protein will be delivered to the apical membrane. If control specimens look like this, data cannot be trusted and the experiment should be repeated with better polarized cells. Note that not all of the expressed to GFP fusion is delivered to the basolateral membrane during the chase of 31 degrees Celsius, as evidenced by the extensive intracellular green signal for the total protein Once mastered, this technique should take approximately 10 to 12 hours After its development.
This technique paved the way for researchers in the field of membrane trafficking to explore regulatory proteins in polarized epithelial cells. After watching this video, you should have a good understanding of how to express proteins in polarized epithelial cells using microinjection technique.
