This is a straightforward method to monitor cellular phenotypes and features of R-pass nuclei. It can be also often recovered in genome-wide study to find the genetic factors affecting the stability of R-pass nuclei. Since is this a well-established motor organism, this technique is easy and does not require much time and effort compared to using human cell lines or animal models.
The aggregation of R-pass nuclei is closely associated with Parkinson's disease. The proteins or chemicals which are likely affecting the stability of R-pass nuclei can be tested in this Humanized yeast model. To begin, patch three single colonies onto an SD-URA agar plate for the selection of cells containing alpha-synuclein plasmids.
Then, inoculate the three-patched yeast transformants per strain into 3 milliliters of SRD-URA for the selective growth of yeast containing the plasmids with 2%raffinose for inhibition of the induction of alpha-synuclein under the Gal1 promoter and 0.1%glucose. Incubate the inoculated culture at 30 degrees celsius under agitation at 200 rotations per minute. The next day, inoculate the overnight cultured cells into 3 milliliters of fresh SRD-URA till they reach an optical density of 0.2 at 600 nanometers.
Then, incubate the culture for six hours at 30 degrees celsius under agitation at 200 rotations per minute. Measure the optical density at 600 nanometer using a spectrophotometer. Next, dilute the samples to an optical density of 0.5 at 600 nanometer with sterile distilled water in lane 1 of a 96-well plate to equalize the number of cells in each culture.
Perform 5-fold serial dilutions of the yeast cells by adding 160 microliters of sterile distilled water to the next five lanes and ensure a total volume of 40 microliters when serially diluting the cells in each lane. Use a multichannel pipette to transfer 10 microliters from each well to spot the samples on SD-URA agar plates for the controls and SG-URA agar plates to monitor toxicity. Incubate the spotted plates upside down at 30 degrees celsius for four days.
Take images of the plates every 24 hours until day four and then analyze the data by comparing the growth differences between strains spotted by eye. Inoculate the three patched yeast transformants per strain into three milliliters of SRD-URA and incubate at 30 degrees celsius under agitation at 200 rotations per minute overnight. The next day, measure the optical density of the cells cultured overnight at 600 nanometers and inoculate the cells into a total of 200 microliters of fresh SD-URA and SG-URA in 96-well plates.
Adjust the final volume including the cells to 200 microliters and adjust the initial cell optical density to 0.05 at 600 nanometers. Adjust the program settings in the microtiter plate. Set the target temperature to 30 degrees celsius, the interval time to 15 minutes, the shaking duration to 890 seconds, the shaking amplitude to 5 millimetres, and the measurement as absorbance at 600 nanometers.
Incubate the plate for 2-3 days for all the strains to reach the stationary phase in a microplate reader. Analyze the data by plotting the growth curve. Inoculate the patched yeast transformants into 3 milliliters of SRD-URA and culture them overnight at 30 degrees celsius under agitation at 200 rotations per minute.
The next day, re-inoculate the overnight cultured cells into 3 milliliters of fresh SRD-URA to an optical density of 0.003 at 600 nanometers, then incubate the culture at 30 degrees celsius under agitation at 200 rotations per minute, until the optical density reaches 0.2. Then add 300 microliters of 20%galactose, and then incubate for another 6 hours at 30 degrees celsius under agitation at 200 rotations per minute. Collect one milliliters of the cell culture by centrifugation at 800 g for 5 minutes and discard the supernatant.
Resuspend the cell pellet gently in 30 microliters of distilled water. Prepare an agarose gel pad on a glass slide. Resuspend the cells and load five microliters of the cell resuspension on the glass slide, and cover the cells using the cut agarose pad for examination.
To perform microscopic imaging with a 100x objective, use the screen capture option for generating images using the program and select brightfield to focus the cell with a 100x objective using immersion oil. Switch to a GFP fluorescence filter and adjust the image exposure time to between 50 millisecond and 300 millisecond to achieve a clear image by balancing the signal-to-noise ratio. Open the image file and analyze the data by counting the cells based on the number of foci in the cells using image analysis software.
The synuclein expression under the Gal1 promoter in the PRS426 vector showed significant growth retardation on the agar plate containing galactose as an inducer. The difference in growth by synuclein expression was also observed in liquid cultures. In both culture conditions, wild-type synuclein and variants with E46K or A53T mutations showed growth defects due to synuclein toxicity.
However, the synuclein A30p variant, which does not form aggregates, showed a non-toxic phenotype. The strains exhibiting severe cytotoxicity showed foci of synuclein aggregates, but in the A30p variant, a less toxic form of synuclein was diffused throughout the cells. To achieve reproducible results, it is important to use the cell in the same growth stage in each experiment, particularly when you monitor synuclein-associated phenotypes.
As we have shown in fluorescent microscope data, synuclein can form larger aggregates. Therefore, it can be simply fractionated by centrifugation and can be quantified by western blotting using synuclein-specific antibody. This technique is applicable for high-throughput screening to find novel genetic factors and chemical compounds affecting the aggregation of synuclein.
Thus, we hope to find new therapeutic candidates for Parkinson's disease.
