Name: studying proteolysis of cyclin b at the single cell level in whole cell populations
Uploaded: 2012-09-17T13:00:00
Description: Watch this Scientific Journal Video about Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations at JoVE.com

Vi er taknemmelige for S. Taylor for at tilvejebringe den pLPCX-histon H2-GFP plasmid. Vi takker R. Mertelsmann til kontinuerlig support. Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft.

1. Såning af U2OS-baserede cyklin B-SNAP Reporter celler (klon 11 Celler 6) på Microscope Chamber Slides <ol><li> Trypsinisér subkonfluente SNAP reporter-celler, der fik lov til at vokse asynkront i logaritmisk fase i mindst 48 timer. </li><li> Arbejde med 8 brønde mikroskop kamre (konstant fordeling af celler). </li></ol> Til podning af celler på 8 brønde mikroskop kamre ved en konstant fordeling over hele overfladen af ​​mikroskopet kammeret, centrifuge 10.000 celler og r...

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	<li>Musacchio, A., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle-assembly+checkpoint+in+space+and+time.">The spindle-assembly checkpoint in space and time.</a> Nat. Rev. Mol. Cell Biol. 8, 379-393 (2007).</li><li>Nasmyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregating+sister+genomes:+the+molecular+biology+of+chromosome+separation.">Segregating sister genomes: the molecular biology of chromosome separation.</a> Science. 297, 559-565 (2002).</li><li>Cahill, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+mitotic+checkpoint+genes+in+human+cancers.">Mutations of mitotic checkpoint genes in human cancers.</a> Nature. 392, 300-303 (1998).</li><li>Lengauer, C., Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+instabilities+in+human+cancers.">Genetic instabilities in human cancers.</a> Nature. 396, 643-649 (1998).</li><li>Fletcher, D. A., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+and+the+cytoskeleton.">Cell mechanics and the cytoskeleton.</a> Nature. 463, 485-4892 (2010).</li><li>Schnerch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+APC/C+activity+in+the+presence+of+chromosomal+misalignment+in+unperturbed+cell+populations.">Monitoring APC/C activity in the presence of chromosomal misalignment in unperturbed cell populations.</a> Cell Cycle. 11, (2012).</li><li>Keppler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+the+covalent+labeling+of+fusion+proteins+with+small+molecules+in+vivo.">A general method for the covalent labeling of fusion proteins with small molecules in vivo.</a> Nat. Biotechnol. 21, 86-869 (2003).</li><li>Jansen, L. E., Black, B. E., Foltz, D. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+centromeric+chromatin+requires+exit+from+mitosis.">Propagation of centromeric chromatin requires exit from mitosis.</a> J. Cell. Biol. 176, 795-805 (2007).</li><li>Malureanu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc20+hypomorphic+mice+fail+to+counteract+de+novo+synthesis+of+cyclin+B1+in+mitosis.">Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis.</a> J. Cell Biol. 191, 313-329 (2010).</li><li>Mena, A. L., Lam, E. W., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+spindle-assembly+checkpoint+response+requires+de+novo+transcription+and+translation+of+cyclin+B1.">Sustained spindle-assembly checkpoint response requires de novo transcription and translation of cyclin B1.</a> PLoS One. 5, (2010).</li><li>Wolthuis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc20+and+Cks+direct+the+spindle+checkpoint-independent+destruction+of+cyclin.">Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin.</a> A. Mol. Cell. 30, 290-302 (2008).</li></ol>

Vi præsenterer her en live-cell imaging tilgang letter samtidig overvågning af cyclin B proteolyse og kromosom tilpasning. Denne tilgang gør det muligt at studere mitotisk kontrol i uperturberede cellepopulationer på enkelt celle niveau. Cyclin B nedbrydning kurver lette direkte indsigt i aktiviteten af APC / C og dermed indirekte afspejler status for SAC 6. Denne fremgangsmåde, selvom fastsat ud fra den Scan ^ R-software, nemt kan reproduceres på nogen sammenlignelig mikros...

<table border="1"><tbody><tr><td> Navn på produkt </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Reporter-cellelinien </td><td> genereret in-house som beskrevet 6 </td><td> klon 11 reporter-celler (U2Os-baserede cyclin B-SNAP udtrykkende celler) </td><td></td></tr><tr><td> Retroviral cyclin B-SNAP ekspressionsvektor </td><td> genereret in-house som beskrevet 6 </td><td> pLNCX2-cyclin B mut5-SNAP </td><td></td></tr><tr><td> Phenolred-free DMEM </td><td> Gibco </td><td> 21063-029 </td><td> Tilskud af FCS, natriumpyruvat, penicillin / strepto-mycin kræves </td></tr><tr><td> SNAP-Cell TMR-Star </td><td> New England Biolabs </td><td> S9105S </td><td> Stamopløsning 400 um i DMSO </td></tr><tr><td> Særlige Opstics Plate, 96 brønde </td><td> Costar </td&gt; <td> 3720 </td><td></td></tr><tr><td> μ-Slide 8 godt, ibiTreat </td><td> Ibidi </td><td> 80.826 </td><td></td></tr><tr><td> Mikroskopi enhed </td><td> Olympus </td><td> IX-81 invers mikroskop med klimakammer </td><td></td></tr><tr><td> Mål </td><td> Olympus </td><td> UPLSAPO 20x objektiv (NA 0,75) </td><td></td></tr><tr><td> Erhvervelse software </td><td> Olympus </td><td> Scan ^ R Acquisition software (v.2.2.09) </td><td></td></tr><tr><td> Analysesoftware </td><td> Olympus </td><td> Scan ^ R Analysis software (v.1.2.0.6) </td><td></td></tr></tbody></table>

studying proteolysis of cyclin b at the single cell level in whole cell populations

Ligelig fordeling af kromosomer mellem de to datterceller ved celledeling er en forudsætning for at sikre genetisk stabilitet 1. Unøjagtigheder i kromosom separation er kendetegnende for malignitet og forbundet med progressiv sygdom 2-4. Spindlen samling checkpoint (SAC) er en mitotisk overvågningsmekanisme, som holder tilbage celler på metafase indtil hver enkelt kromosom har etableret en stabil bipolar tilknytning til mitotiske spindel 1. SAC udøver sin funktion ved interferens med aktiverende APC / C underenhed Cdc20 at blokere proteolyse af Securin og cyclin B og dermed kromosom separation og mitotisk exit. Forkert montering af kromosomer forhindrer silencing af SAC-signalering og forårsager fortsat hæmning af APC / C Cdc20 indtil problemet er løst for at undgå kromosom missegregation, aneuploidi og maligne vækster 1. De fleste undersøgelser, der behandles på influence af forkert kromosomal fastgørelse på APC / C-afhængige proteolyse benyttede sig af spindel forstyrrelser ved hjælp af depolymerisering eller mikrotubulus-stabiliserende medicin til at forstyrre kromosomale tilknytning til mikrotubuli. Da interferens med mikrotubuli kinetik kan påvirke transport og lokalisering af kritiske regulatorer, disse procedurer bærer en risiko for at fremkalde kunstige effekter 5. At undersøge, hvordan SAC interfererer med APC / C-afhængige proteolyse af cyclin B under mitose i uperturberede cellepopulationer, vi etableret et histon H2-GFP-baseret system, der tillod samtidig overvågning af metafase tilpasning af mitotiske kromosomer og proteolyse af cyclin B 6 . At skildre proteolytiske profiler, genererede vi et kimært cyclin B reportermolekyle med en C-terminal SNAP del 6 (fig. 1). I en individuel mærkning reaktion er SNAP-delen i stand til at danne kovalente bindinger medalkylguanin-bærere (SNAP substrat) 7,8 (figur 1). SNAP substratmolekyler er let tilgængelige og bære et bredt spektrum af forskellige fluorochromer. Kimære cyclin B-SNAP-molekyler bliver mærket ved tilsætning af membran-permeable SNAP substrat til vækstmediet 7 (figur 1). Efter mærkningsreaktionen, falder cyclin B-SNAP fluorescensintensitet i et puls-chase reaktion-lignende måde og fluorescensintensiteter afspejler niveauer af cyclin B nedbrydning 6 (fig. 1). Vores system letter overvågningen af ​​mitotiske APC / C-afhængig proteolyse i et stort antal celler (eller flere cellepopulationer) parallelt. Derved kan systemet være et værdifuldt redskab til at identificere agenter / små molekyler, der kan interferere med proteolytisk aktivitet ved metafase til anafase overgangen. Øvrigt, som det syntese af cyclin B under mitose for nylig blevet foreslået som en vigtig mechanism at fremme en mitotisk blok i mus og mennesker ved at holde cyclin B ekspressionsniveauer stabile 9,10, dette system muligt for os at analysere cyclin B proteolyse som et element i en ligevægtssituation 6.

Watch this Scientific Journal Video about Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations at JoVE.com

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Metafase at anafase overgang udløses gennem anaphase-fremmende kompleks (APC / C)-afhængig ubiquitinering og efterfølgende destruktion af cyclin B. Her har vi oprettet et system, som efter puls-chase mærkning, mulighed for at overvåge cyclin B proteolyse i hele cellepopulationer og letter påvisning af indblanding fra mitotisk checkpoint.

Studere Proteolyse af cyclin B på enkelt celle niveau i hele cellepopulationer

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

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