The overall goal of the following experiment is to quantitatively measure the sub nuclear organization in yeast cells and how it's affected by changes in growth conditions or mutations. This is achieved by growing yeast cells to log phase in conditions that will minimize autofluorescence. Next, the cells are placed into a growth chamber for viewing under the microscope.
Then images are taken to measure the distance between different fluorescently labeled structures in the cell. Results are obtained that show differences in non-nuclear organization, which is dependent on changing the nutritional conditions during growth of the yeast culture. This method can help answer key questions in the epie field about the interplay between subar organization or chromatin and gene regulation.
Toul fish and yeast begin by preparing Edinburgh minimal medium or EMM and EMM with also ammonium chloride to reduce autofluorescence rather than autoclave. Use a 0.2 micron filter to sterilize the glucose solution and add it to the autoclave media. Inoculate three milliliters of EMM in 30 milliliter tubes with fish and yeast cells.
Freshly grown on an agar plate in the rich medium, YEA lightly cap the tubes and incubate the cells in a shaker at 225 RPM and 30 degrees Celsius. Keep the cells in log phase for two days by counting them with a birka chamber every morning and evening and diluting them to the appropriate density. On the day of the experiment.
The cells should be an early log phase to nitrogen staff. The cells refer to the procedure outlined in the written protocol to collect the cells in one pellet at the bottom of a tube centrifuge, 1.5 milliliters of cells in an eend orph tube at a maximum of 1.5 RCF by first spinning the tube for two minutes, then turning it 180 degrees for an additional two minute spin. Remove all but 10 to 15 microliters of the supernatant and resuspend the cells in the remaining medium, tho an aliquot of one milligram per milliliter filter sterilized lectin.
Add five microliters of lectin solution to the corner of the cover glass and add five microliters of culture to the lectin drop. To create a growth chamber of S pom base cells, pipette five microliters of EMM on a clean slide and invert the cover glass with the culture at a 70 degree angle over the medium and drop it top down onto the EMM to seal the edges with silicon grease. Cut the wide end of a 200 microliter tip and attach the narrow end to a two milliliter syringe.
Fill the syringe with approximately one milliliter of silicon grease. Apply a fine line of silicon to the edges of the cover glass by gently pushing the piston of the syringe to image the growth chamber. Begin by using Brightfield Imaging to locate the cells and dig.
Get a sharp picture for confocal microscopy. We use a Zeiss LSM 700 laser scanning microscope with a plan apoch chromat 63 times oil objective lens, and a 16 line average plane scan setting. Set the area units to one to 1.1 to obtain an optical slice of 0.8 microns.
Scan the cells with lasers that detect Alexa 4 88 and mCherry or GFP. Record enough images to take measurements in 60 cells for each drain. Switch to a new growth chamber with fresh cells.
After 60 minutes of imaging, open an image in the Zeen Light Edition program to measure the distance between signals of different fluorophores in the same focal plane. Open the measurements tool and adjust the light intensity and contrast to identify the center of each fluorescent signal, for example, the spindle pole body and nuclear membrane, and measure the distance between them. Transfer the data to a notepad sheet.
Compare the mean or median subcellular distances between different strains and treatments using statistical software or tests such as the T-test or the Man Whitney Rank sum test. In this example strain, PJ 1 18 5 was grown in EMM and a sample was placed into a growth chamber for imaging. Then an aliquot of PJ 1 18 5.
Culture was transferred into EMMN and incubated for 15 minutes before a sample was placed into a growth chamber for imaging. The measuring tool was used to measure the distance between the locus and the spindle pole body as well as between the locus and the nuclear membrane. As shown here, there was a statistically significant difference in the distance of the gene cluster moving away from the nuclear membrane towards the spindle pole body in the nitrogen starved cells compared to cells grown in nitrogen.
The data measuring the distance between the GFP and the SBP had a normal distribution, and hence the mean distances could be compared using a T-test. There was a significant difference between the two mean values. The data measuring the distance between the GFP and NM did not have a normal distribution, and hence the median distances were compared using a man Whitney ranked sum test.
There was a significant difference between the two median values Following this procedure. Other methods, like real-time PCR can be performed in order to answer additional questions on how the gene expression level correlates to the changes in sub nuclear organization.
