The overall goal of this Time-Lapse Video Microscopy is to track the selective Parkin-mediated removal of damaged mitochondria during the mitophagy process. This method can help answer key questions in the molecular metabolism field, such as how mitophagy's affect enduring pathological conditions. The main advantage of this technique is that it provides a very powerful tool for following the dynamics of labeled proteins during their molecular processes.
This method can provide insight into the mitochondrial life cycle. It can also be applied to other systems, such as primary cell cultures, embryology studies, and predicting an priority. The immortalized mouse embryonic fibroblasts used in this study are grown in complete DMEM medium on a ten centimeter tissue culture plate at 37 degrees Celsius in a humidified atmosphere containing five percent carbon dioxide.
When the cells are at 80 percent confluency, discard the medium by sterile suction and add ten milliliters of sterile PBS. Remove the PBS, and add one milliliter of 0.25 percent Trypsin EDTA. Incubate the plate at 37 degrees Celsius for two to three minutes until the cells are detached.
Add four milliliters of complete DMEM medium, and re suspend the cells. Take out ten microliters of the cell suspension for counting with a hemocytometer. Seed one times ten to the sixth cells on to a ten centimeter tissue culture dish, and incubate for 24 hours.
On the following day, wash the cells once with PBS and detach the cells by trypsinization, as demonstrated earlier. Add four milliliters of complete DMEM medium, re-suspend the cells, and transfer the cell suspension to a 15 milliliter tube. Spin the cells at 250 gs and four degrees Celsius for five minutes.
Discard the supernatant and re suspend the pellet in one milliliter of sterile PBS. Spin the cells again. Discard the supernatant after the second spin.
Add 100 microliters of the solution mix for electroporation to the cell pellet and gently re suspend the pellet by pipetting. Then add to the cell suspension two micrograms of EYFP Parkin expression plasmid and one microgram of DsRed2 Mito expression plasmid. Use the disposable Pasteur pipette to transfer the solution to a sterile cuvette.
Electroporate the cells using a preset program. Immediately after the electroporation, add 500 microliters of fresh, pre warmed complete DMEM medium to the cells and seed the cells on a six centimeter. live-imaging grade tissue culture plate.
Incubate the cells in a humidified atmosphere containing five percent carbon dioxide at 37 degrees Celsius for 24 hours. Prior to starting this procedure, set the temperature of the microscope's chamber at 37 degrees Celsius. Next, prepare a specific live imaging medium by mixing the following, Phenol-free DMEM supplemented with ten percent FBS, two millimolar per liter of L-Glutamine, 100 units per milliliter of penicillin, and 100 milligrams per milliliter of streptomycin.
Pre warm the medium at 37 degrees Celsius in a water bath. Discard the medium of the electroparated cells by sterile suction and add one mililiter of pre warmed live imaging medium. Incubate the plate for 30 minutes at 37 degrees Celsius.
After confirming that the microscope's chamber is at a stable temperature of 37 degrees Celsius. Influx five percent carbon dioxide into the chamber. Carefully, and without major movements or oscillations, place the plate with the electroporated cells, into the microscope's chamber.
Using the software interface, set the microscope to detect the fluorescent signals from the fusion proteins encoded by the EYFP Parkin and DsRed2 Mito expression vectors. Open the time lapse video microscopy software. In the upper menu, select FITC for EYFP Parkin and Rhodamine for DsRed2 Mito.
Select a magnification of 20X. Using the software interface, look for a single cell expressing both cotransfected vectors and register the position. Do this for a minimum of 10 cells for every experimental condition.
Select the menu, Apps and click on, Multi Dimensional Acquisition. In the Multi Dimensional Acquisition windows, select the parameters needed, such as the number of acquisitions, the interval of time between each acquisition, and the position of the recorded cell. Click on, Acquire to start the basal acquisition of both fluorescent signals.
Collect images every five minutes for a total interval of fifteen minutes. While image acquisition is occurring, prepare pre warmed live imaging medium containing Carbonyl Cyanide 4 trifluouromethoxy phenylhydrazone, or FCCP, at a concentration twice the final working concentration. Interrupt the acquisition process, and gently add one milliliter of the pre warmed live imaging medium with FCCP into the plate inside the microscope's chamber.
After adding FCCP, it is important to re focus the microscope before restarting the image acquisition. Restart the acquisition process as previously described, and collect images for a total period of three hours using the recorded position. When image acquisition is complete, save all the acquired images for later analysis.
Shown here are time lapse images of EYFP-Parkin and DsRED2-Mito fluorescent signals in fibroblasts prior to FCCP administration. The white boxes represent the magnified areas of the respective panels on the right. During the basal time points, EYFP-Parkin, visualized in green, is homogeneously diffused throughout the cell.
The mitochondria network, visualized in red, appears to be well interconnected, as indicated by the white arrows. Following FCCP administration, mitochondria fractionation due to membrane depolarization is observed at five minutes. However, EYFP-Parkin is still homogeneously diffused throughout the cytoplasm.
At 55 minutes post FCCP administration, EYFP-Parkin is recruited to the damaged mitochondrial membranes to trigger the mitophagy process. The movement of EYFP-Parkin to the mitochondrial membrane following FCCP administration is shown in this representative time lapse movie. Once mastered, this technique can be done in two hours if it is performed properly.
After its development, this technique paved the way for researchers in the field of autophagy experiment mitochondrial recycling. Allowing cells to study the mitophagy process as a mitochondrial quality control in the cells.
