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

एस टेलर को pLPCX Histone H2 GFP प्लाज्मिड प्रदान करने के लिए आभारी हैं. हम निरंतर समर्थन के लिए आर Mertelsmann धन्यवाद. यह काम ड्यूश Forschungsgemeinschaft द्वारा समर्थित किया गया.

1. माइक्रोस्कोप चैंबर स्लाइड पर U2OS आधारित Cyclin बी SNAP रिपोर्टर कोशिकाओं (क्लोन 11 प्रकोष्ठों 6) की सीडिंग <ol><li> Trypsinize subconfluent SNAP संवाददाता कोशिकाओं है कि asynchronously लॉग चरण में कम से कम 48 घंटे के लिए विकसित करने के लिए अ?...

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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>

हम यहाँ एक जीवित कोशिका इमेजिंग आधारित दृष्टिकोण cyclin बी proteolysis और गुणसूत्र संरेखण के साथ - साथ निगरानी की सुविधा मौजूद है. इस दृष्टिकोण की अनुमति देता है mitotic में नियंत्रण के अध्ययन एकल कोशिका के स्तर पर सेल ...

<table border="1"><tbody><tr><td> उत्पाद का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> संवाददाता सेल लाइन </td><td> उत्पन्न घर में 6 के रूप में वर्णित </td><td> 11 रिपोर्टर कोशिकाओं क्लोन (U2Os आधारित बी तस्वीर cyclin व्यक्त कोशिकाओं) </td><td></td></tr><tr><td> रेट्रोवायरल cyclin बी SNAP अभिव्यक्ति वेक्टर </td><td> उत्पन्न घर में 6 के रूप में वर्णित </td><td> pLNCX2 cyclin बी mut5-SNAP </td><td></td></tr><tr><td> Phenolred मुक्त DMEM </td><td> Gibco </td><td> 21063-029 </td><td> FCS, सोडियम पाइरूवेट, पेनिसिलिन / strepto Mycin के साथ पूरकता की आवश्यकता </td></tr><tr><td> तस्वीर सेल TMR-स्टार </td><td> न्यू इंग्लैंड Biolabs </td><td> S9105S </td><td> DMSO में स्टॉक समाधान 400 सुक्ष्ममापी </td></tr><tr><td> विशेष Opstics प्लेट, अच्छी तरह से 96 </td><td> Costar </td&gt; <td> 3720 </td><td></td></tr><tr><td> 8 μ स्लाइड अच्छी तरह से ibiTreat, </td><td> Ibidi </td><td> 80826 </td><td></td></tr><tr><td> माइक्रोस्कोपी इकाई </td><td> ओलिंप </td><td> IX-81 जलवायु चैम्बर के साथ उलटा माइक्रोस्कोप </td><td></td></tr><tr><td> उद्देश्य </td><td> ओलिंप </td><td> UPLSAPO 20x उद्देश्य (0.75 NA) </td><td></td></tr><tr><td> अधिग्रहण सॉफ्टवेयर </td><td> ओलिंप </td><td> स्कैन ^ आर अधिग्रहण सॉफ्टवेयर (v.2.2.09) </td><td></td></tr><tr><td> विश्लेषण सॉफ्टवेयर </td><td> ओलिंप </td><td> स्कैन ^ आर विश्लेषण सॉफ्टवेयर (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

संक्रमण Anaphase Anaphase जटिल (APC / सी) पर निर्भर ubiquitination और cyclin बी के बाद यहाँ विनाश को बढ़ावा देने, हम एक प्रणाली है, जो नाड़ी - पीछा लेबलिंग के बाद स्थापित, पूरे सेल आबादी में cyclin बी proteolysis निगरानी की अनुमति देता है और के माध्यम से शुरू हो रहा है मेटाफ़ेज़ mitotic जांच की चौकी से हस्तक्षेप का पता लगाने की सुविधा है.

पूरे सेल आबादी में एकल कोशिका के स्तर पर Cyclin बी के proteolysis अध्ययन

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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