Name: spatiotemporal analysis of cytokinetic events in fission yeast
Uploaded: 2017-02-20T14:00:00
Description: Watch this Scientific Journal Video about Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast at JoVE.com

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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C., 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=The+(1,3)beta-D-glucan+synthase+subunit+Bgs1p+is+responsible+for+the+fission+yeast+primary+septum+formation.">The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation.</a> Mol Microbiol. 65 (1), 201-217 (2007).</li><li>Cortes, J. C., 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=Cooperation+between+Paxillin-like+Protein+Pxl1+and+Glucan+Synthase+Bgs1+Is+Essential+for+Actomyosin+Ring+Stability+and+Septum+Formation+in+Fission+Yeast.">Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.</a> PLoS Genet. 11 (7), e1005358 (2015).</li><li>Cortes, J. C., Ishiguro, J., Duran, A., Ribas, J. 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. C., Sullivan, W., Karess, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessing+the+role+and+dynamics+of+nonmuscle+myosin+II+during+furrow+formation+in+early+Drosophila+embryos.">Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos.</a> Mol Biol Cell. 15 (2), 838-850 (2004).</li><li>Figard, L., Xu, H., Garcia, H. G., Golding, I., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasma+membrane+flattens+out+to+fuel+cell-surface+growth+during+Drosophila+cellularization.">The plasma membrane flattens out to fuel cell-surface growth during Drosophila cellularization.</a> Dev Cell. 27 (6), 648-655 (2013).</li><li>Wei, B., 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=Unique+Spatiotemporal+Activation+Pattern+of+Cdc42+by+Gef1+and+Scd1+Promotes+Different+Events+during+Cytokinesis.">Unique Spatiotemporal Activation Pattern of Cdc42 by Gef1 and Scd1 Promotes Different Events during Cytokinesis.</a> Mol Biol Cell. , (2016).</li><li>Nabeshima, K., 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=Dynamics+of+centromeres+during+metaphase-anaphase+transition+in+fission+yeast:+Dis1+is+implicated+in+force+balance+in+metaphase+bipolar+spindle.">Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.</a> Mol Biol Cell. 9 (11), 3211-3225 (1998).</li><li>Johnson, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dma1+ubiquitinates+the+SIN+scaffold,+Sid4,+to+impede+the+mitotic+localization+of+Plo1+kinase.">Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase.</a> EMBO J. 30 (2), 341-354 (2011).</li><li>Yonetani, 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=Regulation+of+cytokinesis+by+the+formin+cdc12p.">Regulation of cytokinesis by the formin cdc12p.</a> Curr Biol. 20 (6), 561-566 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fission Yeast: A laboratory Manual. , (2016).</li><li>Hachet, O., Simanis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid1p/anillin+and+the+septation+initiation+network+orchestrate+contractile+ring+assembly+for+cytokinesis.">Mid1p/anillin and the septation initiation network orchestrate contractile ring assembly for cytokinesis.</a> Genes Dev. 22 (22), 3205-3216 (2008).</li><li>Zhou, Z., 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=The+contractile+ring+coordinates+curvature-dependent+septum+assembly+during+fission+yeast+cytokinesis.">The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis.</a> Mol Biol Cell. 26 (1), 78-90 (2015).</li><li>Chen, B. 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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 ...

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