This work was supported by startup funds from The University of Tennessee and TN-SCORE, a multi-disciplinary research program sponsored by NSF-EPSCoR (EPS-1004083).

1. Preparation of Sample
<ol>
	<li>Grow fission yeast cells expressing the spindle pole body marker Sad1-mCherry23 and type II myosin regulatory light chain Rlc1-Tomato24 in YE liquid media at 32 &#176;C for 8 generations. 
	NOTE: For temperature sensitive mutants, grow cells at 25 &#176;C. Grow cells to mid-log phase to an O.D.600 of 0.5 in YE. For more on fission yeast growth conditions refer to the Fission yeast lab manual25.</li...

<ol>
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A., Minc, N., Boudaoud, A., Chang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+turgor+pressure,+the+contractile+ring,+and+septum+assembly+to+forces+in+cytokinesis+in+fission+yeast.">Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast.</a> Curr Biol. 22 (17), 1601-1608 (2012).</li><li>Munoz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+cell+wall+beta(1,3)glucan+is+required+to+couple+septation+to+actomyosin+ring+contraction.">Extracellular cell wall beta(1,3)glucan is required to couple septation to actomyosin ring contraction.</a> J Cell Biol. 203 (2), 265-282 (2013).</li><li>Wang, N., Lee, I. J., Rask, G., Wu, J. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+(1,3)beta-D-glucan+synthase+catalytic+subunit+homologue+Bgs1p/Cps1p+from+fission+yeast+suggests+that+it+is+involved+in+septation,+polarized+growth,+mating,+spore+wall+formation+and+spore+germination.">Localization of the (1,3)beta-D-glucan synthase catalytic subunit homologue Bgs1p/Cps1p from fission yeast suggests that it is involved in septation, polarized growth, mating, spore wall formation and spore germination.</a> J Cell Sci. 115 (Pt 21), 4081-4096 (2002).</li><li>Liu, J., Tang, X., Wang, H., Oliferenko, S., Balasubramanian, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+the+integral+membrane+protein+Cps1p+to+the+cell+division+site+is+dependent+on+the+actomyosin+ring+and+the+septation-inducing+network+in+Schizosaccharomyces+pombe.">The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe.</a> Mol Biol Cell. 13 (3), 989-1000 (2002).</li><li>Royou, A., Field, C., Sisson, J. 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The authors declare no competing financial interests.

Here we have described a protocol to study cytokinetic events in fission yeast in a temporal manner. The protocol described here provides temporal resolution of different cytokinetic events with reference to each other; timing of protein recruitment or loss with reference to cytokinetic phase; structure of the ring throughout the different phases of cytokinesis; and the progression of cytokinesis with reference to mitosis. With this protocol it is possible to precisely define the cytokinetic phase that may be altered in ...

Cytokinesis, the final step in cell division, is a complex process essential for proper differentiation, development, and survival of an organism. Cytokinesis involves multiple events that are organized to ensure successful cell separation while maintaining genomic integrity1. Cytokinesis involves events where an actomyosin ring once assembled undergoes constriction, which is concurrent with membrane expansion and furrowing, and extracellular cellular matrix remodeling, finally followed by cell abscission1,2,3. Improper organization of cytokinetic events can lead to cell separation and ploidy defects, and may cause diseases like cancer4,5,6,7,8. The fundamental principles that enable organization of cytokinetic events are not well understood, thus leading to roadblocks in our understanding of the etiology of these diseases.
The fission yeast Schizosaccharomyces pombe (S.&#160;pombe)&#160;is an excellent model system to study cytokinesis due to the conserved nature of the proteins involved1. In fission yeast, after actomyosin ring assembly, the ring enters a maturation/dwell phase where it does not constrict9. Maturation ends with initiation of actomyosin ring constriction, concurrent with membrane furrowing and septum ingression. Seminal work over the years have given us a fairly good understanding of actomyosin ring assembly in fission yeast1,9,10. In some eukaryotes, including fission yeast, successful assembly of the actomyosin ring is not sufficient for membrane furrowing. In fission yeast, ring constriction alone does not provide sufficient force to overcome internal turgor pressure for furrow formation11. A recent model indicates that this force is instead provided by septum ingression11. In another model, the role of plasma membrane extension has been suggested to contribute to furrow formation12,13. Ring constriction and membrane furrowing do not occur in Bgs1/Cps1 temperature sensitive mutant cps1-191, the major enzyme for primary septum formation14,15. Cells lacking Bgs1 show defective primary septum and prolonged ring constriction15,16. Bgs1 is recruited to the cell division site for septum ingression during maturation after actomyosin ring assembly17,18. Similarly, during cellularization in Drosophila embryos, ring constriction is biphasic with a significantly slow initial constriction rate19, resembling the maturation phase observed in fission yeast. Biphasic ring constriction may slow down membrane furrowing to allow for sufficient membrane expansion20 and modification of extracellular matrix. This suggests that after actomyosin ring assembly, ring constriction occurs efficiently only when the cell has satisfied the requirements for furrow formation. It is not well understood what conditions are required for actomyosin ring constriction after ring assembly, nor the molecular events that regulate this process. We have recently shown that following actomyosin ring assembly the small GTPase Cdc42 undergoes a unique spatiotemporal activation pattern21. This pattern is established by the unique localization pattern of the Cdc42 guanidine nucleotide exchange factors (GEFs) that activate Cdc42. The GEF Gef1 localizes to the assembled actomyosin ring and promotes onset of ring constriction and septum ingression, while Scd1 localizes to the furrowing membrane and promotes normal septum formation. We find that the Cdc42 activation pattern established by its GEFs lead to the regulation of distinct cytokinetic events.
To understand the molecular mechanism of events that eventually lead to cell separation following actomyosin ring assembly, one needs to follow the distinct cytokinetic events in time and space. In fission yeast, cytokinesis first involves the assembly of precursor nodes around the nucleus, which eventually recruit the type II myosin, the formin Cdc12, and other proteins required for actomyosin ring assembly. To time the distinct cytokinetic events and provide a frame of reference, the separation of spindle pole body markers (spindle formation) is considered as time 022. The assembly of the actomyosin ring can be followed by monitoring over time the intensity of a fluorescently tagged actomyosin ring protein such as the type II myosin regulatory light chain Rlc1. Here we describe a microscopic approach to analyze different stages of cytokinesis over time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast extract media</td>
			<td>Sunrise Science Products</td>
			<td>YES 225</td>
			<td>0.5% w/v yeast extract, 3% w/v glucose, 225mg/L adenine, histidine, leucine, uracil, and lysine</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem LE agarose, Lonza</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottomed culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Coverslip No. 1.5 was used. This will vary as per the microscope specifications used.&#160;</td>
		</tr>
		<tr>
			<td>VT-Hawk 2D array laser scanning confocal microscopy system</td>
			<td>Visitech International, UK</td>
			<td></td>
			<td>with an Olympus IX-83 inverted microscope with a 100X / numerical aperture 1.49 UAPO lens (Olympus) and EM-CCD digital camera (Hamamatsu).&#160;</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

spatiotemporal analysis of cytokinetic events in fission yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

Fission yeast cells expressing the ring marker, Rlc1-GFP (green, Figure 2) and spindle pole body marker Sad1-mCherry (red, Figure 2) were imaged during cytokinesis. Onset of spindle pole body marker (white arrow, Figures 2A, B) is considered as time 0. Rlc1-GFP signal appears at time -4 min with reference to spindle pole body separation (red arrow, Figures 2A, 2B). Rlc1-GFP signal forms a continuous ring 10 min post spindle pole body sepa...

Watch this Scientific Journal Video about Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast at JoVE.com

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

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.

spatiotemporal-analysis-of-cytokinetic-events-in-fission-yeast

Research

JoVE Journal

Biology

7.3K Views.  University of Tennessee. 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.T...