The overall goal of the following experiment is to follow the uptake of mitochondria into the vae as a means of studying the autophagy of mitochondria, a process known as mitophagy. This is achieved by expressing in yeast cells, a genetically encoded and mitochondrial targeted dual emission fluorescence, pH biosensor named Mt Rosea that labels mitochondria with red and green fluorescence. As a second step, yeast cells are subjected to nitrogen starvation in order to induce autophagy.
Next, we view cells by fluorescence microscopy or confocal laser scanning microscopy in order to visualize uptake of mitochondria into the va. Results are obtained that show accumulation of red but not green fluorescence in the VAs of nitrogen starved cells. Indicative of autophagy scoring of cells in a population allows the degree of mitophagy to be determined.
Using rosea to monitor the uptake of mitochondria into the vacuole of yeast cells can provide mechanistic insights into the process of mii. The approach can be adapted to monitor the autophagy of other cellular compartments such as the nucleus demonstrating the procedure will be dalibor alica, a postdoctoral researcher from our laboratory. In this protocol we use wild type strain BY 4 7 41, and a deletion mutant strain delta A TG three, derived from the same genetic background To visualize the localization of mitochondria.
In these strains, they are labeled with a fluorescent reporter. Rosea Rosea is a dual color emission biosensor comprising a relatively pH stable red fluorescent protein and a pH sensitive green fluorescent protein. The mitochondrial target sequence is used to target the Rosea biosensor to the mitochondrial matrix.
This reporter has been named Mt.Rosea at high pH the biosensor fluoresces both red and green. However, at low pH it fluoresces only red. The wild type strain is a control to indicate the levels of autophagy normally expected as a negative control.
A strain null for the expression of an essential autophagy gene is suitable. Since such a strain cannot deliver mitochondria to the vacuole, standard procedures are used to transform the yeast strains with the plasmid carrying the mitochondrial targeted rosala reporter growth plates are incubated for two to four days at 28 to 30 degrees Celsius until the appearance of colonies about two to three millimeters in diameter to grow cells for induction of autophagy, inoculate for each strain, a single yeast colony into 10 milliliters of growth. Medium containing a non fermentable carbon source.
In this case, ethanol and the appropriate ox atrophic supplements. Duplicate cultures are grown for each strain resulting in a total of four cultures. Incubate yeast cultures for approximately 48 hours at 28 to 30 degrees Celsius with orbital shaking by which time the cultures should be in the mid log phase of growth.
At the end of the incubation period, transfer two one milliliter samples of each culture into 1.7 milliliters. Snap cap plastic centrifuge tubes gently pellet the cells by centrifugation at low speed. After centrifugation, carefully remove and discard the supernatant from each tube without disturbing the cell pellet.
Wash the cells with one milliliter of sterile distilled water by resuspension, followed by centrifugation. Repeat the wash two more times to remove residual medium. After the third wash, resuspend the cells in either 100 microliters of growth, medium or nitrogen starvation.
Medium nitrogen starvation medium contains the carbon source ethanol, but lacks nitrogen supplements and will induce autophagy in cells. Next, re inoculate the resuspended cells into 10 milliliters of fresh growth. Medium or 10 milliliters of nitrogen starvation.Medium.
Incubate the cultures with orbital shaking for six to 12 hours after the induction of autophagy. It is useful to confirm the delivery of Mt Rosea to the vae under the fluorescence microscope. The vae can be located by labeling using a cumerin based blue fluorescent dye, such as seven amino four chloro methyl cumerin, L arginine mi cmac arg for short.
Labeling with cmac ARG does not need to be performed routinely, but it is recommended for those who are not familiar with the location and appearance of the vacuole in yeast. To begin the procedure for labeling the vacuole transfer one milliliter samples of each culture into separate 1.7 milliliters snap cap plastic centrifuge tubes. Then add cmac arg to each tube.
Incubate the tubes at 28 to 30 degrees Celsius with orbital shaking for 30 minutes. After 30 minutes, pellet the cells by centrifugation, discard the supernatant, wash the cells with one milliliter of sterile distilled water by suspension, followed by centrifugation. Wash the cells two more times with sterile distilled water.
This step removes residual medium and excess cmac arg die. After the third wash, resus suspend the cells in 100 microliters of sterile distilled water. The cells are now ready to be mounted for imaging by fluorescence microscopy.
When carrying out this protocol, it's very important to use freshly transformed yeast cultures to ensure you obtain T cells with highly fluorescent mitochondria. To prepare live yeast cells for imaging by fluorescent microscopy, first melt two milligrams of low melting point aeros in separate one milliliter aliquots of growth medium and nitrogen starvation medium by incubating at 70 degrees Celsius for one hour, the molten aros is maintained at 70 degrees Celsius. To mount the cells spot 20 microliters of molten aros onto a labeled 76 by 26 millimeter glass microscope.
Slide immediately spot 10 microliters of the washed cell suspension onto the aros bed. Cover the aros bed with a 22 by 22 millimeter glass cover slip. The cells will spread across the agros surface beneath the cover slip.
Then carefully seal the edges of the cover slip with a thin film of nail polish. This will prevent dehydration and movement of the cover slip During imaging when the nail polish is completely dry, the slides are ready to be viewed by fluorescence microscopy cells mounted. Using this technique can be observed for up to one hour after mounting without any apparent adverse effects on cells.
Since yeast cells are relatively small, you will need a good quality fluorescence microscope equipped with excitation emission filters suitable for the separate visualization of GFP emission and RFP emission and a 60 x water immersion objective lens with a numerical aperture of 1.2. Typical results obtained using MT Rosea expressed in wild type cells and using confocal laser scanning. Microscopy are shown here under non starved conditions with ethanol as a carbon source while type cells exhibit a typical cellular distribution of mitochondria at the periphery of the yeast cells that show both red and green fluorescence.
Red and green fluorescence emission is not detected in the vacuole. When MT.Rosea expressing wild type cells are subjected to nitrogen starvation for six hours and beyond. They exhibit in addition to red and green fluorescence corresponding to mitochondria the accumulation of red but not green fluorescence in the va.
This ular signal is indicative of autophagy. The autophagic uptake of the mitochondria ular. Localization of rosea can be separately confirmed using CMAC arg.
In contrast, mitophagy negative control cells expressing MT.Rosea observed before starvation and following six hours and beyond of starvation show no accumulation of red fluorescence in the vacuole. While mitochondrial labeling remains strong to assess the level of autophagy in the wild type and A TG three mutant strains, greater than 200 cells per strain were observed for accumulation of red fluorescence in the ole before the induction of autophagy and at selected time points Following induction of autophagy plotting the proportion of cells showing ular accumulation reveals that the degree of ular uptake increases with time in wild type cells, but not in delta ATG three mutant cells that cannot deliver mitochondria to the vacuole After its development. This technique pa the way for researchers in the field of autophagy to explore the delivery of mitochondria and other organelles such as the nucleus to the VA and the various growth conditions in these cells.
