The overall goal of this live cell microscopy technique is to study to the spatiotemporal events that occur during cytokinesis in the fission yeast model system. This method can help answer key questions in the cytokinetic field such as the molecular details of the different stages of cytokinesis over time. The main advantage of this technique is that different spatial components of the cytokinetic machinery can be analyzed over long periods of time with minimal toxicity to the cells.
Though this method can provide insight into cytokinesis and fission yeast, it can also be applied to budding yeast and other fungi. To begin this procedure, pour three to four milliliters of melted media laced with Vitamin C onto a glass-bottomed culture dish. Once the media has solidified, use a sharp scalpel to gently raise the media slab from the culture dish, and place a pipette tip between the media slab and the culture dish to prevent the slab from falling back into the dish.
Next load two to five microliters of re-suspended cell culture between the media slab and the glass bottom of the culture dish. Then very gently remove the pipette tip and place the media slab back in its original position on the culture dish. Incubate the cells in the culture dish for 30 minutes to an hour in the dark at the temperature the microscopy will be performed.
When the cells are ready for imaging, place a drop of wild on the outer side of the glass on the culture dish. Place the culture dish on an inverted microscope. Focus on the medial plane of the cells, and ensure that they are well-spaced and not too crowded.
Make sure to start image acquisition of the cells in a suitable stage of the cell cycle. In this experiment, it is late G2.Next, program the image acquisition software. Set the exposure time for DIC to 100 milliseconds.
For GFP and RFP filters, use exposure times of 75 milliseconds. Set the laser power to 50%but note that this will vary depending upon the individual experiment. To capture images in three dimensions, program the software to acquire Z series.
Use of a maximum step size of 4 micrometers and a distance of 3.0 micrometers around the central focus point of the cells for the capture of 16 Z frames per time point for the total Z distance of 6 micrometers. These parameters can be altered in accordance with the thickness of the cell. Set the program to take images every two minutes.
Select the exposure time based on the requirements of the experiment. In this example, 75 milliseconds is used. Program the software to stop acquisition after 90 minutes.
And then start the image acquisition. ImageJ software is used to analyze the image. To begin, open the image series for each of the wavelengths.
Select a cell to be studied. Double click on the line option of the toolbar to open another window to adjust the line width. Adjust the line width to correspond to the cell width which is about 40 to 50 pixels for a wild type cell.
Draw a line along the long axis of the cell of interest. Click on analyze then tools then ROI manager and then click on add. This will add the selected line to the ROI window for later use.
Do not close this window. Click on the image of interest then select edit, then selection, then straighten. Check process entire stack to straighten the cell horizontally.
Click on image then transform then rotate 90 degrees right to straighten the image vertically. Click on image then stacks then make montage. This will open a window for assigning the number of rows and columns for the stack, the scale factor for the image size, the first and last slice or frame for the montage, and the frame increment for the montage.
Check label slices and hit enter. As a window with a montage of the cell of interest is shown, open the image series for the other wavelength. In the ROI manager, click on the line identifier to select the same cell on the second image file.
Repeat the previous steps to open a montage of the second image. On the image with the spindle pole body marker, Sad1-mCherry, look for the separation of the spindle pole body marker, and mark that time point as time zero. Follow the signal of the ring protein Rlc1-tomato on the image over time, and look for the signal that appears as a distinct line as opposed to the patches of Rlc1-tomato.
This time point marks completion of actomyosin ring assembly or the start of the maturation phase. Next scroll through the movie over time to determine when the Rlc1-tomato ring starts to decrease in size. This is marked as the end of maturation phase or onset of ring constriction.
Follow the Rlc1-tomato signal over time throughout constriction until it appears as a dot in the middle of the cell axis. This is marked as the end of ring constriction or end of septum ingression. Following the montage of DIC images, determine when abscission is completed.
Record the time point when the cells physically separate. Begin this analysis by selecting a cell with a visible actomyosin ring. Double click on the line option of the ImageJ toolbar.
Adjust the line width to correspond to the ring thickness about 15 to 20 pixels. Draw a line along the ring, taking care to ensure the entire thickness of the ring is included. Click on analyze, then tools, then ROI Manager, and click on add.
This will add the select line to the ROI window for later use. Do not close this window. Click on the image of interest and select edit, then selection, then straighten.
Check process entire stack to straighten the ring horizontally. Reset the intensity of the brightest plane in the straighten Z stack using image, then adjust, then brightness and contrast, then reset. Click on the tab SGK then 3D project to open a dialogue box.
Change the projection method to brightest point and slice spacing to two to three pixels. Finally, change the axis of rotation to X or Y-axis depending upon the desired viewing angle. Click interpolate and click OK to generate a 3D projection of the image.
Use the scroll bar beneath the image to rotate the image. Next, open the image series for the other wavelength. Click on the line identifier in the ROI manager to select the same ring on the second image file.
Repeat the previous steps to open a 3D ring in the image with the other wavelength. With both 3D image rings open, click on image, then color, then merge channels. Select each image from the dropdown menu for the corresponding color desired and click OK.Compare the localization of different markers with regards to the localization at the cytokinetic ring or ingressing membrane.
Fission yeast cells expressed in the spindle pole body marker, Sad1-mCherry and ring marker Rlc1-GFP were imaged during cytokinesis. Spindle pole body separation occurred at 17 minutes, designated as time zero. Rlc1-GFP appears at the division site four minutes before spindle pole body separation.
After spindle pole body separation, ring maturation starts at ten minutes. Ring constriction begins at 31 minutes, and ends at 53 minutes. Finally, cell abscission occurs at 80 minutes after spindle pole body separation.
The timeline of cytokinetic events determined in the previous images can be plotted in a graph with reference to mitosis as determined by the distance between the spindle pole body markers. Analysis of the ring marker Rlc1-tomato and the septum membrane marker Bgs1-GFP at the division site throughout cytokinesis reveal that the ring assembled before Bgs1-GFP recruitment. As the ring constricts, Bgs1-GFP also localizes to the ingressing membrane adjacent to the constricting ring.
Finally, Rlc1-tomato signal dissipates after ring constriction while Bgs1-GFP appears as a disk, remaining within the membrane barrier. The efficiency of cytokinetic events can be determined by the distribution of proteins along the ring. Here Cdc15-GFP distribution along the ring is even on the left image but uneven on the right image.
Once mastered, this technique can be done in two to three hours if performed properly. While attempting this procedure, it's important to remember to choose cells with an appropriate field, ideally containing six to eight well-spaced cells in late G2 or early M phase, with no visible impurities in the agar. After watching this video, you should have a good understanding of how to spatiotemporally analyze protein localization during cytokinesis.
