The overall goal of this method is to monitor the entire sexual reproduction process in fission yeast by light microscopy. This method describes the preparation of fission yeast cells for time-lapse imaging of various stages of the sexual lifecycle, such as mating partner choice, polarized growth, cell-cell fusion, myosis, and sporulation. The main advantage of this technique is that it provides single cell visualization and so overcomes the problem that cells do not enter sexual differentiation synchronously.
This method is very simple, but the visual demonstration is useful to show how to mount several strains in parallel for microscopy. Demonstrating the procedure with my PhD student Omaya Dudin, will be Laura Merlini, a postdoc in my lab. There are several variations to this protocol which can be tailored to study various stages of the sexual lifecycle, using either homothallic or heterothallic strains.
In this demonstration, homothallic H90 strains will be used. In these strains, mating type switching takes place during the last mitotic division after nitrogen deprivation, resulting in a mix of the two mating types. In the evening, two days prior to the mating experiment, inoculate freshly streaked strains from solid media into culture tubes containing 3 milliliters of minimum sporulation medium with nitrogen or MSL plus N.Incubate overnight with shaking at 25 degrees Celsius.
On the following morning, if necessary, dilute the cell suspensions in MSL plus N to ensure exponential growth throughout the day. In the evening, measure the optical density and dilute the cells in 20 milliliters of MSL plus N media in 100 milliliter flasks to an optical density of 025 Wild-type cell cultures should reach an optical density 600 of about 8 the following morning. So the dilution should be adjusted accordingly, if working with strains with longer generation times.
Incubate the cultures overnight at 30 degrees Celsius with shaking. On the following morning, measure the optical density to verify that each culture has an optical density 600 around 8. Pellet the cells at 1000 x g for one minute and transfer it to a 1.5 milliliter tube.
Wash the cells three times in 1 milliliter of minimum sporulation medium without nitrogen or MSL minus N.After the last wash, resuspend the cells in 3 milliliters of MSL minus N medium. Measure the optical density and dilute the cells to an optical density 600 of 1.5. To begin the preparation of agarose pads, first melt an Aliquat of MSL minus N agarose 2 percent in a 95 degree Celsius heat block for 10 to 15 minutes.
In the meantime, pellet 100 microliters of the cell suspension prepared earlier at 1000 x g for one minute. Remove the supernatant and resuspend cells in the residual 2 to 4 microliters of medium. With the cells waiting on the bench, add 200 microliters of the melted medium on a glass slide between two spacers.
Gently place another slide on top of the melted agar. Once the agarose has solidified, carefully remove the spacers. To remove the top glass slide, gently rotate and pull it sideways.
If several strains are to be imaged simultaneously, the pad can be subdivided into four mini pads. For this, use a razor blade and split the pad in its middle pulling the parts about 1 millimeter aside and repeat in the perpendicular direction. Add 1 microliter of cells to the agarose pad and wait for about one minute.
Cover the cells with a cover slip and seal the entire periphery of the cover slip with heated VELAP. It is important to seal the entire cover slip to prevent desiccation of the pad. Let the pad sit for at least 30 minutes before imaging.
To ensure success of the protocol, and high mating efficiency, cells are to be kept in exponential growth until nitrogen starvation and mounted without excess liquid so they are stationary on the agarose pad. Live cell imaging of the mating yeast cells is then performed at 25 degrees Celsius, or room temperature, following the procedure described in the protocol text. Time-lapse imaging allows a visualization of cells undergoing shmooing, fusion, and sporulation in the 24 hours that follow nitrogen starvation.
This process is illustrated in this representative movie by the two cells magnified in the bottom-left inset. This protocol can also be used to monitor the dynamics of fluorescently tagged proteins in mating cells. Here, a heterothallic H-strain expressing myo52 tagged with 3GFP was mated with a heterothallic H+strain expressing myo52 fused to the red fluorescent tdTomato protein, as well as cytosolic GFP.
The cytosolic GFP allows monitoring of the timing of fusion, visualized by its entry into the H-cell. These time-lapse images show that myo52 initially formed to dynamic zones throughout the cell cortex. Myo52 signal was then stabilized in a single focus just before the fusion event.
After watching this video, you should have a good understanding of how to prepare cells for efficient mating and live cell investigation of the mating process of fission yeast.
