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. Forberedelse af prøven <ol><li> Grow fission gærceller der udtrykker spindelpol-legemer markør Sad1-mCherry 23 og type II myosin regulatoriske let kæde Rlc1-Tomato 24 i YE flydende medier ved 32 ° C i 8 generationer. BEMÆRK: temperaturfølsomme mutanter, vokser celler ved 25 ° C. Grow celler til midten logaritmisk fase til en OD 600 på 0,5 i YE. For mere om fission betingelser gær vækst refererer til fission gær lab manual <sup class="xref...

<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. 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Her har vi beskrevet en protokol til at studere cytokinetic begivenheder i fission gær i en tidsmæssig måde. Den her beskrevne protokol giver tidsmæssige opløsning af forskellige cytokinetic begivenheder med henvisning til hinanden; timing af protein rekruttering eller tab med henvisning til cytokinetic fase; struktur af ringen i de forskellige faser af cytokinese; og progression af cytokinesen med henvisning til mitose. Med denne protokol er det muligt præcist at definere den cytokinetic fase, der kan ændres i f...

Cytokinese, det sidste trin i celledeling, er en kompleks proces med henblik på forsvarlig differentiering, udvikling og overlevelse af en organisme. Cytokinese involverer flere hændelser, der er organiseret til at sikre en vellykket celleseparation samtidig bevare genomisk integritet 1. Cytokinese involverer arrangementer, hvor en actomyosin ring er samlet undergår konstriktion, som er sideløbende med membran ekspansion og furer, og ekstracellulær matrix remodellering endelig efterfulgt af celle abscission 1, 2, 3. Forkert organisering af cytokinetic hændelser kan føre til celle separations- og ploidi defekter, og kan forårsage sygdomme som kræft 4, 5, 6, 7, 8. De grundlæggende principper, der muliggør organization af cytokinetic begivenheder er ikke godt forstået, hvilket fører til vejspærringer i vores forståelse af ætiologien af ​​disse sygdomme. Den fission gær Schizosaccharomyces pombe (S. pombe) er en fremragende model til undersøgelse cytokinese grundet den konserverede beskaffenhed af de involverede 1 proteiner. I fission gær, efter actomyosin ring samling, ringen ind i en modning / dvæle fase, hvor det ikke snøre 9. Modning slutter med initiering af actomyosin ring konstriktion, samtidig med membran furer og septum indtrængen. Seminal arbejde gennem årene har givet os en rimelig god forståelse for actomyosin ring forsamling i fission gær 1, 9, 10. I nogle eukaryoter, herunder fission gær, vellykket samling af actomyosin ring er ikke tilstrækkelig til membran furer. I fission gær, ring konstriktion alene giver ikke tilstrækkelig kraft til at overvinde intern saftspændingen for fure dannelse 11. En nylig model indikerer, at denne kraft i stedet leveres af septum indtrængen 11. I en anden model, har den rolle, plasmamembranen udvidelse blevet foreslået at bidrage til fure formation 12, 13. Ring konstriktion og membran furer ikke forekommer i Bgs1 / CPS1 temperaturfølsom mutant cps1-191, de store enzym til primær septum dannelse 14, 15. Celler, der mangler Bgs1 viser defekt primære septum og langvarig ring konstriktion 15, 16. Bgs1 rekrutteres til celledeling site for septum indtrængen under modning efter actomyosin ring samling 17, 18. Similarly, under cellularization i Drosophila embryoer, ring konstriktion er bifasisk med en signifikant langsom indledende konstriktion sats 19, ligner modningsfasen observeret i fission gær. Bifasisk ring konstriktion kan bremse membran furer at give mulighed for tilstrækkelig membran udvidelse 20 og ændring af ekstracellulær matrix. Dette antyder, at efter actomyosin ring samling, ring konstriktion sker effektivt, når cellen har opfyldt kravene for fure formation. Det er ikke godt forstået hvilke betingelser er nødvendige for actomyosin ring konstriktion efter ringsamlingen, eller de molekylære begivenheder, der regulerer denne proces. Vi har for nylig vist, at følgende actomyosin ring forsamling den lille GTPase Cdc42 gennemgår en unik Spatiotemporal aktivering mønster 21. Dette mønster er etableret ved den unikke lokalisering mønster af cdc42 guanidin nukleotid exchange faktorer (GEFs), der aktiverer Cdc42. GEF Gef1 lokaliseres til den samlede actomyosin ring og fremmer indtræden af ​​ring konstriktion og septum indtrængen, mens SCD1 lokaliseres til furer membran og fremmer normal septum formation. Vi finder, at Cdc42 aktivering mønster indført ved dens GEFs fører til regulering af forskellige cytokinetic begivenheder. For at forstå den molekylære mekanisme af begivenheder, der i sidste ende fører til celleseparation efter actomyosin ringsamlingen, er man nødt til at følge de forskellige cytokinetic begivenheder i tid og rum. I fission gær, cytokinese først involverer samling af precursor-knudepunkter omkring kernen, som i sidste ende rekrutterer type II myosin, den formin Cdc12, og andre proteiner, der er nødvendige for actomyosin ringaggregat. Til tid distinkte cytokinetic begivenheder og giver en referenceramme, er adskillelsen af spindel pole krop markører (spindel dannelse) betragtes som tiden 0 22. Samlingen af ​​the actomyosin ring kan følges ved overvågning over tid intensiteten af ​​en fluorescens mærkede actomyosin ring protein, såsom type II myosin regulatoriske let kæde Rlc1. Her beskriver vi en mikroskopisk fremgangsmåde til at analysere forskellige stadier af cytokinese over tid.

<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

Cytokinese, det sidste skridt i celledeling er afgørende for at opretholde genom integritet. Korrekt cytokinese er vigtig for celledifferentiering og udvikling. Cytokinese involverer en række begivenheder, der er godt koordineret i tid og rum. Cytokinese involverer dannelsen af ​​en actomyosin ring i division site, efterfulgt af ring konstriktion, membran fure dannelse og ekstracellulær matrix remodellering. Den fission gær, Schizosaccharomyces pombe (S. pombe) er en velundersøgte modelsystem, der har afsløret med betydelig klarhed de indledende begivenheder i cytokinese. Men vi forstår ikke helt, hvordan forskellige cytokinetic begivenheder koordineres spatiotemporally. For at afgøre dette, er man nødt til at analysere de forskellige cytokinetic begivenheder i store detaljer i både tid og rum. Her beskriver vi en mikroskopi tilgang til at undersøge forskellige cytokinetic evenser i levende celler. Med denne fremgangsmåde er det muligt at tid forskellige cytokinetic begivenheder og bestemme tidspunktet for rekruttering af forskellige proteiner under cytokinese. Desuden beskriver vi protokoller til at sammenligne proteinlokalisering og distribution på stedet for celledeling. Dette er en grundlæggende protokol for at studere cytokinese i fission gær og kan også anvendes til andre gær og fungale systemer.

Fission gærceller der udtrykker ringen markering, blev Rlc1-GFP (grønt, figur 2) og spindelpol-legemer markør Sad1-mCherry (rød, figur 2) afbildes under cytokinese. Indtræden af spindelpol-legemer markør (hvid pil, figur 2A, B) betragtes som tid 0. Rlc1-GFP signal vises på tidspunkt -4 minutter med henvisning til spindelpol-legemer separation (rød pil, figur 2A, 2B). Rlc1-GFP signal danner en kontinuerlig ring 10 min efter spinde...

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

Den fission gær, Schizosaccharomyces pombe er en fremragende model til undersøgelse cytokinese, den sidste fase i celledeling. Her beskriver vi en mikroskopi tilgang til at analysere forskellige cytokinetic begivenheder i levende fission gærceller.

Spatiotemporal Analyse af Cytokinetic Begivenheder i Fission Gær

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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Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Spatiotemporal Kortlægning af Motilitet i<em&gt; Ex vivo</em&gt; Forberedelser af tarmene

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Microscopy of Fission Yeast Sexual Lifecycle

Mikroskopi af fission Gær Seksuel Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Anvendelser af pHluorin Kvantitative, Kinetic og High-throughput Analyse af Endocytose i Spirende gær

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Høj opløsning Imaging og analyse af de enkelte Astral mikrotubulusdynamik i Spirende Gær

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

4D-mikroskopi af gær

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Undersøgelse af mitotisk og meiotisk fission gær nuklear dynamik ved fluorescens levende celle mikroskopi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Kvantificering af spatiotemporale parametre for cellulær exocytose i mikropatterned celler

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...