By synchronizing cells in prophase one and then depleting the target protein at a particular stage, this method can identify temporarily distinct functions of specific proteins. Conditionally depleting proteins at specific stages of myosis prevents the residual effects a traditional myotic mutant can have on later stages. This method could be used to study budding yeast mitosis and other organisms that do not undergo nuclear envelope breakdown.
In addition, this method can be used in other organisms to study cell cycle events prior to nuclear envelope breakdown. To begin, make an agar pad that will be used to create a monolayer of cells for imaging. Cut off and discard the cap and bottom one third of a 1.5-milliliter microcentrifuge tube to create a cylinder that will serve as a mold for the agar pad.
Place the cut microcentrifuge tube cylinder on a clean glass slide with the top of the tube upside down, sitting on the slide. Make six milliliters of a 5%agar solution in a 50-milliliter beaker. Cut off the tip of the pipette to make a larger opening and pipette approximately 500 microliters of melted agar into the microcentrifuge tube.
Let it sit at room temperature until the agar solidifies. To prepare yeast cells, spin down 200 microliters of the sporulation culture at 800 G for two minutes in one-milliliter microcentrifuge tubes. Discard 180 microliters of the supernatant.
Resuspend the pellet in the remaining supernatant by swirling and flicking the tube. Pipette six microliters of the concentrated cells onto the cover slip in the middle of the chamber on the spot with ConA. Hold the cylinder with the agar pad and carefully slide it off the glass slide.
Make sure the agar is completely flat at the bottom and using the bottom of the pipette tip, apply a slight amount of pressure to the microcentrifuge mold such that the agar pad is pushed out slightly above the boundary of the tube. Next, invert the mold such that the agar pad is facing down towards the chamber. Use forceps to gently place the agar pad on top of the cells and the pipette tip to gently slide the agar pad around the chamber 10 to 20 times to create a monolayer of cells on the cover slip.
Keep the agar pad in the chamber for 12 to 15 minutes. Next, transfer two milliliters of sporulation culture to two microcentrifuge tubes and spin at 15, 700 G for two minutes. After transferring the supernatant into clean microcentrifuge tubes, spin again at the same condition and collect the supernatant to clean microcentrifuge tubes.
Add two milliliters of the supernatant dropwise to the chamber containing the agar pad. Once the liquid has reached the top of the chamber, the agar pad will most likely float. Remove the agar pad gently with forceps and discard it.
Place a 24 by 50 millimeter cover slip on the top of the chamber to prevent evaporation during imaging. To set up the movie on the microscope, fit the cover slip inside the slide holder. Adhere the molding clay to the side of the cover slip to keep it secure in the slide holder.
Open the image acquisition software. Use coarse and fine adjustment knobs to focus the cells using DIC or bright field. On the main menu of the image acquisition software, click on File, select Acquire, and four windows will pop up.
In the window named Resolve 3D, click on the Erlenmeyer flask icon. This will open a window titled Design Run Experiment, which contains the controls for setting up an experiment to set up a time-lapse movie. Under the Design tab, navigate to the tab labeled Sectioning.
Select the box next to Z sectioning and set the optical section spacing to one micrometer and the number of optical sections to five. Next, under the Channels tab, click on the plus icon and select the appropriate channel. Then, select the box next to Reference Image and set the Z position to the middle of the sample from the dropdown menu.
Select a value of transmittance and exposure time from the dropdown menu. Under the Time-lapse tab, select the box next to Time-lapse and enter values for minutes and hours to identify the time interval of image acquisition. Select the box next to Maintain Focus with Ultimate Focus to prevent stage drift during the movie.
And under the Points tab, select the box next to Visit Point List. In the main menu, click on View and select Point List. Move the stage to an area of the chamber that shows a monolayer of cells.
Click on Mark Point in the Point List window. Move the stage to select 25 to 30 points without any overlap to avoid overexposing the cells and image each field during each time course. In the Point List window, select Calibrate All to set ultimate focus for each point.
Under the Run tab, save the file to the appropriate destination on the computer. On the Run Experiment window, select the Play button to start the movie. Open the Fiji software.
Open the DIC and mCherry channels. Obtain a single maximum intensity projection of the mCherry channel by clicking on Image, followed by Stacks and Z Project and select Max Intensity from the dropdown menu. To merge the DIC and mCherry channels in one image, click on Image, Color, and select Merge Channels.
Follow a single cell through meiosis. After meiosis II completion, record the number of DNA masses. In wild type cells with the anchor away background, there are typically four DNA masses at the end of meiosis II, representing the four products of meiosis.
In a small fraction of the cells, only three masses are visible after meiosis II.When Ctf19-FRB is anchored away at the time of release of prophase I, approximately 47%of cells display more than four DNA masses upon the completion of meiosis, suggesting a defect in the attachment of kinetochores and microtubules. With anchoring away of Ctf19-FRB, either after kinetochore assembly but before meiosis I, or after meiosis II, approximately 16%of cells display additional DNA masses. One of the most important parts of this protocol is to add the supernatant dropwise to your chamber to avoid disrupting your monolayer.
Additionally, note the time required to prepare for imaging to consider it in your analysis. By changing fluorescently packed proteins, this procedure can be used to answer the questions regarding cell cycle duration, chromosome segregation, protein abundance, and protein localization.
