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

Мы благодарны С. Тейлор за предоставление pLPCX-гистонов H2-GFP плазмиды. Мы благодарим Р. Mertelsmann за постоянную поддержку. Эта работа была поддержана Deutsche Forschungsgemeinschaft.

1. Посев из U2OS на основе циклин B-SNAP Reporter Cells (клон 11 ячеек 6) на предметные стекла палата <ol><li> Trypsinize субконфлюентные SNAP репортер клетки, которые могут расти асинхронно в логарифмической фазе, по крайней мере, 48 часов. </li><li> Работа с 8 хорошо камер микроскопа (постоянное распределение ...

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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>

Мы приведем здесь живой клетке изображений подход, основанный на содействие одновременного мониторинга циклин B протеолиза и хромосомы выравнивания. Такой подход позволяет исследовать митотический управления в невозмущенной клеточных популяций на одном уровне клетки. Cyclin кривые B д?...

<table border="1"><tbody><tr><td> Наименование продукта </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> Линии Reporter клетки </td><td> генерируется в доме, как описано 6 </td><td> клонировать 11 клеток Репортер (U2OS на основе циклин B-клеток, экспрессирующих SNAP) </td><td></td></tr><tr><td> Ретровирусные циклин B-SNAP вектор экспрессии </td><td> генерируется в доме, как описано 6 </td><td> pLNCX2-циклин B mut5-SNAP </td><td></td></tr><tr><td> Phenolred без DMEM </td><td> Gibco </td><td> 21063-029 </td><td> Дополнение с FCS, пируват натрия, пенициллина / стрепто-MYCIN требуется </td></tr><tr><td> SNAP-Cell TMR-Star </td><td> New England Biolabs </td><td> S9105S </td><td> Со 400 мкМ раствор в ДМСО </td></tr><tr><td> Специальные пластины Opstics, 96 а </td><td> Costar </td&gt; <td> 3720 </td><td></td></tr><tr><td> μ-Slide 8 хорошо, ibiTreat </td><td> Ibidi </td><td> 80826 </td><td></td></tr><tr><td> Микроскопия блок </td><td> Олимп </td><td> IX-81 обратного микроскоп с климатической камере </td><td></td></tr><tr><td> Цель </td><td> Олимп </td><td> UPLSAPO 20x цели (NA 0,75) </td><td></td></tr><tr><td> Приобретение программного обеспечения </td><td> Олимп </td><td> Сканирование ^ R Приобретение программного обеспечения (v.2.2.09) </td><td></td></tr><tr><td> Анализ программного обеспечения </td><td> Олимп </td><td> Сканирование ^ R анализа программного обеспечения (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

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 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

Метафазы к анафазе переход срабатывает через анафазе способствующих комплекса (APC / C)-зависимых убиквитинирования и последующее уничтожение циклин B. Здесь мы создали систему, которая после импульсно-погоня маркировки, позволяет контролировать протеолиза циклин B во всей популяции клеток и облегчает обнаружение вмешательства митотической контрольно-пропускном пункте.

Изучение Протеолиз циклин B на одном уровне клеток в популяции клеток Всего

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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