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

我们非常感谢S.泰勒提供的pLPCX组蛋白H2-GFP质粒。我们感谢R. Mertelsmann为持续的支持。这项工作是由德国研究联合会的支持。

1。显微镜玻片上播种的U2OS细胞周期蛋白B-SNAP记者细胞（克隆11个单元6） <ol><li> Trypsinize汇合的的SNAP记者被允许异步增长对数生长期至少48小时的细胞，。 </li><li>与8孔显微镜室（恒定细胞分布）。 </li></ol>用于播种的细胞到8以及显微镜室在一个恒定的分布在整个表面的显微镜室，离心10,000个细胞，并重新悬浮在350μl的无酚红的正常生长培养基（补充有10％牛胎儿血清，...

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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>

我们在座的活细胞成像为基础的方法，促进细胞周期蛋白B同时监测蛋白质和染色体排列。这种方法允许在单细胞水平上研究的有丝分裂控制中泰然自若的细胞群。细胞周期蛋白B降解曲线的APC / C的活性和促进直接的见解，从而间接地反映了状态的SAC 6。 这种方法，即使建立了基于扫描^ R软件，可以很容易地被复制任何类似的显微镜站。此外，使用我们的逆转录病毒的?...

<table border="1"><tbody><tr><td> 产品名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>报道细胞系</td><td>产生的房子，6 </td><td>克隆11个记者细胞 （U2OS基于细胞周期蛋白B-SNAP细胞） </td><td></td></tr><tr><td>逆转录病毒的细胞周期蛋白B-SNAP表达载体</td><td>产生的房子，6 </td><td> pLNCX2细胞周期蛋白B mut5-SNAP </td><td></td></tr><tr><td>免费的phenolred-DMEM </td><td> Gibco公司</td><td> 21063-029 </td><td> FCS，丙酮酸钠，青霉素/链霉素补充要求</td></tr><tr><td> SNAP-细胞TMR明星</td><td> New England Biolabs公司</td><td> S9105S </td><td>原液400μM的在DMSO中</td></tr><tr><td>特别Opstics板，96孔</td><td>深圳科诗特</td&gt; <td> 3720 </td><td></td></tr><tr><td> μ-幻灯片8，ibiTreat </td><td> Ibidi </td><td> 80826 </td><td></td></tr><tr><td>显微镜装置</td><td>奥林巴斯</td><td> IX-81倒置显微镜与气候室</td><td></td></tr><tr><td>目标</td><td>奥林巴斯</td><td> UPLSAPO 20X物镜（NA 0.75） </td><td></td></tr><tr><td>采集软件</td><td>奥林巴斯</td><td>扫描^ R采集软件（v.2.2.09） </td><td></td></tr><tr><td>分析软件</td><td>奥林巴斯</td><td>扫描^ R分析软件（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

两个子细胞在细胞分裂过程中染色体之间平等分配的前提条件是保证遗传稳定性1。染色体分离过程中的不准确之处是恶性肿瘤的一个标志，并伴有渐进性疾病2-4。有丝分裂纺锤体组装检查点（SAC）是一个监督机制，阻碍直到每一个染色体的细胞在中期建立了稳定的双极连接到有丝分裂纺锤体1。 SAC发挥其功能的干扰与激活APC / C亚基CDC20阻止蛋白水解的securin和细胞周期蛋白B和染色体分离和有丝分裂退出。不当安装的染色体防止沉默的SAC的信号，导致继续抑制APC / C CDC20，直到问题得到解决，以避免染色体分离现象的原因，非整倍体和恶性生长。 在我的大多数研究APC / C-依赖的蛋白降解的影响及的不当染色体附件上利用了主轴中断使用微管解聚或稳定的药物来干扰微管与染色体附件。由于干扰微管动力学影响的运输和国产化的关键调节，这些程序承担的风险，导致人为影响5。 要研究如何SAC干扰APC / C-依赖的蛋白降解的细胞周期蛋白B的有丝分裂过程中泰然自若的细胞群，我们建立了组蛋白H2-GFP为基础的系统，该系统允许同时监测中期调整的有丝分裂染色体的细胞周期蛋白B 6和蛋白水解。 要描绘的蛋白水解的公司，我们产生了嵌合细胞周期蛋白B，报道分子的C-末端的SNAP部分6（ 图1）。 SNAP-部分是在一个自标记反应，能够形成共价键烷基鸟嘌呤-载体（SNAP基板）7,8（ 图1）。 SNAP底物分子都是现成的，进行广泛的不同的荧光染料。嵌合细胞周期蛋白B-SNAP分子成为膜可渗透SNAP衬底的生长介质7（ 图1）添加后标记。荧光强度的细胞周期蛋白B-SNAP标记反应后，在一个脉冲追踪反应类似的方式和荧光强度反映细胞周期蛋白B降解6（ 图1）的水平的下降。我们的系统有利于在大量的细胞（或多个细胞群）在平行的有丝分裂，APC / C-依赖的蛋白降解的监测。由此，该系统可以是一个有价值的工具，以确定代理/小分子，能够干扰蛋白水解活性，在中期到后期过渡。此外，作为在有丝分裂过程中合成的细胞周期蛋白B最近已被建议作为一个重要mechanis米，在小鼠和人类保持cyclin B的表达水平基本稳定9,10促进有丝分裂块，这个系统使我们能够分析细胞周期蛋白B蛋白水解的一个元素平衡的平衡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

中期向后期过渡被触发，通过后期促进复合物（APC / C）依赖的泛素化和随后的破坏细胞周期蛋白B.在这里，我们建立了一个系统，脉冲追踪标签，可以监控在整个细胞群的细胞周期蛋白B蛋白水解和有利于检测干扰的有丝分裂关卡。

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

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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