Name: detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay
Uploaded: 2017-06-09T15:00:00
Description: Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

Guikema实验室的研究由荷兰科学研究组织（VIDI授权016126355）和“Stichting Kinderen Kankervrij”KiKA（项目252）的创新研究奖励计划资助。

1.细胞治疗和DNA损伤诱导<ol><li>在补充有20％FCS，50μMβ-巯基乙醇，2mM L-谷氨酰胺，100U / mL青霉素和100μg/ mL链霉素的IMDM中培养人BCR-ABL + B细胞急性成淋巴细胞细胞系BV173或SUP-B15在5％CO 2的气氛中37℃。在6孔板中以5mL /孔计数细胞并以2×10 6细胞/ mL平板。 注意：任选地，可以使用各种来源的悬浮细胞系。 </li><li> 为了阻止细胞周期G1期的BCR-ABL + B-ALL细胞，加入5μM的伊马替尼?...

<ol>
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Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+checkpoint+and+repair+machineries+at+DNA+damage+sites.">Assembly of checkpoint and repair machineries at DNA damage sites.</a> Trends Biochem Sci. 35 (2), 101-108 (2010).</li><li>Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+DNA+double-strand+break+repair+foci+in+mammalian+cells.">Assembly and function of DNA double-strand break repair foci in mammalian cells.</a> DNA Repair. 9 (12), 1219-1228 (2010).</li><li>Matsuoka, S., 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=ATM+and+ATR+substrate+analysis+reveals+extensive+protein+networks+responsive+to+DNA+damage.">ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.</a> Science. 316 (5828), 1160-1166 (2007).</li><li>Barlow, C., Brown, K. D., Deng, C. X., Tagle, D. A., Wynshaw-Boris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+selectively+regulates+distinct+p53-dependent+cell-cycle+checkpoint+and+apoptotic+pathways.">Atm selectively regulates distinct p53-dependent cell-cycle checkpoint and apoptotic pathways.</a> Nature Genet. 17 (4), 453-456 (1997).</li><li>Tibbetts, R. S., 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=A+role+for+ATR+in+the+DNA+damage-induced+phosphorylation+of+p53.">A role for ATR in the DNA damage-induced phosphorylation of p53.</a> Genes Dev. 13 (2), 152-157 (1999).</li><li>Schermelleh, L., 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=Subdiffraction+Multicolor+Imaging+of+the+Nuclear+Periphery+with+3D+Structured+Illumination+Microscopy.">Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</li><li>Chapman, J. R., Sossick, A. J., Boulton, S. J., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1-associated+exclusion+of+53BP1+from+DNA+damage+sites+underlies+temporal+control+of+DNA+repair.">BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.</a> J Cell Sci. 125 (Pt 15), 3529-3534 (2012).</li><li>Gullberg, M., 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=A+sense+of+closeness:+protein+detection+by+proximity+ligation.">A sense of closeness: protein detection by proximity ligation.</a> Curr Opin Biotechnol. 14 (1), 82-86 (2003).</li><li>S&#246;derberg, O., 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=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45 (3), 227-232 (2008).</li><li>Dong, Y., Shannon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+immunosensing+using+antigen+and+antibody+monolayers+on+gold+surfaces+with+electrochemical+and+scanning+probe+detection.">Heterogeneous immunosensing using antigen and antibody monolayers on gold surfaces with electrochemical and scanning probe detection.</a> Anal Chem. 72 (11), 2371-2376 (2000).</li><li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-damage+response+in+human+biology+and+disease.">The DNA-damage response in human biology and disease.</a> Nature. 461 (7267), 1071-1078 (2009).</li><li>Economopoulou, P., Pappa, V., Papageorgiou, S., Dervenoulas, J., Economopoulos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+DNA+repair+mechanisms+in+common+hematological+malignancies.">Abnormalities of DNA repair mechanisms in common hematological malignancies.</a> Leuk Lymphoma. 52 (4), 567-582 (2011).</li><li>Rendleman, J., 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=Genetic+variation+in+DNA+repair+pathways+and+risk+of+non-Hodgkin's+lymphoma.">Genetic variation in DNA repair pathways and risk of non-Hodgkin's lymphoma.</a> PloS One. 9 (7), e101685 (2014).</li><li>Ochodnicka-Mackovicova, K., 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+DNA+Damage+Response+Regulates+RAG1/2+Expression+in+Pre-B+Cells+through+ATM-FOXO1+Signaling.">The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.</a> J Immunol. , (2016).</li><li>Banin, S., 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=Enhanced+phosphorylation+of+p53+by+ATM+in+response+to+DNA+damage.">Enhanced phosphorylation of p53 by ATM in response to DNA damage.</a> Science. 281 (5383), 1674-1677 (1998).</li></ol>

在本报告中，证明PLA可用于确定和可视化悬浮细胞培养物中蛋白质之间的特异性相互作用。值得注意的是，这里描述的方案不限于DNA修复复合物的研究，而且还适用于可视化和定量悬浮细胞培养物中的其他蛋白质 - 蛋白质相互作用。显示当暴露于DNA损伤诱导剂时，ATM激酶与G1-阻滞的BCR-ABL + B-ALL细胞中的磷酸化p53相互作用。以前，我们使用PLA技术来表明ATM和FOXO1转录因子在BCR-ABL + B-ALL细胞中相互作?...

DNA损伤触发高度受限制的一系列事件，涉及蛋白质 - 蛋白质相互作用和翻译后修饰，可确保DNA的有效快速修复，从而保护基因组完整性1 。通常，通过（共聚焦）荧光显微镜监测所谓的电离辐射诱导病灶（IRIF）的形成，研究暴露于电离辐射的细胞中的DNA修复。许多DNA修复和DNA损伤感应蛋白形成IRIF，其代表在染色质位点成核的蛋白质复合物，其维持DNA损伤2,3 。随着时间的推移，IRIF的位置和分辨率为DNA修复的时空组织提供了重要的见解，并且可能表明不同DNA修复途径的参与。损伤的DNA损伤和细胞周期阶段的性质决定了哪个DNA修复途径被激活。佛<html> r实例，在积极参与DNA复制（S期）的细胞中，同源重组（HR）是显性DNA修复途径，而在细胞周期的G1-或G2 / M期细胞中，同源末端连接（NHEJ）修复途径占优势。 DNA损伤后的最早事件之一是激活DNA损伤感应激酶共济失调血管扩张 - 突变蛋白（ATM），其主要在细胞周期的G1-和G2 / M期活跃并调节NHEJ，和共济失调血管扩张症和Rad3相关蛋白（ATR），其通过激活HR作用于S期。 ATM和ATR都是非常多效的激酶，磷酸化参与DNA修复，细胞死亡和存活的许多蛋白质4 。已经显示两种激酶在暴露于基因毒性应激后磷酸化并激活肿瘤抑制蛋白p53，表明这些激酶是关键肿瘤抑制信号轴的上游介质“xref”&gt; 5，6 。 IRIF的形成和组成通常通过使用双色免疫荧光染色和显微镜测定不同蛋白质的共定位来评估，然而，并非所有作为修复蛋白复合物的蛋白质形成IRIF，这限制了该方法的适用性。此外，（共焦）免疫荧光显微镜受光衍射性质的限制，导致约200-300nm的相当差的空间分辨率，超过大多数亚细胞结构的大小，其基本上禁止蛋白质 - 蛋白质相互作用的直接询问分子水平。因此，通过（共焦）荧光显微镜检测的免疫荧光染色模式的共定位不一定表示直接的蛋白质 - 蛋白质相互作用。最近，已经开发了新的超分辨率技术，如三维结构照相显微镜（3D-SIM） 7 ，成功应用于纳米尺度细胞研究53BP1和BRCA1 IRIF形成，揭示了共聚焦激光扫描显微镜无法检测到的这些蛋白质的空间分布特征。 可以使用其他几种方法来检测体内的蛋白质 - 蛋白质相互作用，例如共免疫沉淀，下拉法和酵母双杂交筛选方法。然而，这些技术相当繁琐，需要大量的细胞或蛋白质或涉及蛋白质的过表达，其引入实验工件。最近，已经开发了一种新技术，其允许原位 （ 即在细胞和组织中）的蛋白质 - 蛋白质相互作用的可视化和定量，称为邻近连接测定（PLA） 9,10 。镨通过与寡核苷酸（所谓的PLA探针）缀合的二级抗体检测识别感兴趣的两种蛋白质的重要​​抗体。如果两种不同的二级抗体由于被第一抗体识别的蛋白质之间的相互作用而足够接近，则缀合的寡核苷酸杂交并连接形成封闭的环状DNA底物。随后通过滚环扩增扩增此圆形底物，并用荧光染料缀合的互补寡核苷酸显色。使用PLA，蛋白质 - 蛋白质相互作用的亚细胞定位被保留，因为荧光标记的滚环扩增产物保持附着于PLA探针。基于抗体直径约为7-10nm 11的发现，该测定法的分辨率&lt;50nm。只有两对抗体（primary + secon）才能进行滚环扩增dary）在由其大小（10 + 10 + 10 + 10 = 40nm）定义的周界内物理相互作用。信号放大步骤增加了PLA测定的灵敏度，并且能够检测到几乎不表达的蛋白质的相互作用。 PLA产生可以在每个细胞基础上量化的点状，类似焦点的信号模式，由此可以评估蛋白质 - 蛋白质相互作用中的细胞内和细胞间变化。 DNA修复复合物和IRIFs的形成和组成主要在贴壁细胞系如人骨肉瘤上皮细胞系U2OS，人胚胎肾细胞系HEK293和视网膜色素上皮细胞系RPE-1中进行研究，生长和易转染。使用诸如淋巴样和骨髓细胞系的悬浮细胞培养物不太频繁，因为它们不易于转染，并且通常不粘附到盖玻片上，因此需要额外的/替代的eps成像。然而，DNA损伤的解决方案在淋巴样和骨髓恶性肿瘤的情况下非常相关，因为DNA损伤反应经常受到这些肿瘤中的基因组（驱动）畸变的影响，在正常淋巴和骨髓的恶性转化中起关键作用（祖细胞） 12,13,14 。 该协议描述了如何在悬浮细胞培养物中诱导DNA损伤后，如何使用PLA来评估和定量蛋白质 - 蛋白质相互作用。在这里，执行PLA以确定和可视化在诱导经历G1期细胞周期停滞的人B细胞白血病细胞中的DNA损伤时ATM和p53之间的相互作用。值得注意的是，本文提出的方案并不限于研究在G1停滞的白血病细胞中的ATM和p53相互作用，而且还可以用于其他蛋白质 - 蛋白质相互作用在各种细胞类型和悬浮细胞培养物中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BV173 cell line</td>
			<td>DSMZ</td>
			<td>AC-20</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>SUP-B15 cell line</td>
			<td>DSMZ</td>
			<td>ACC-389</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td>lot #: 064M3396</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco (Life Technologies)</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>imatinib methanesulfonate</td>
			<td>LC Laboratories</td>
			<td>I-5508</td>
			<td>Dissolve in DMSO, prepare 10 mM stock solution</td>
		</tr>
		<tr>
			<td>neocarzinostatin</td>
			<td>Sigma Aldrich</td>
			<td>N9162</td>
			<td>Mutagenic/teratogenic, handle with care</td>
		</tr>
		<tr>
			<td>KU55933</td>
			<td>Selleckchem</td>
			<td>S1092</td>
			<td>Dissolve in DMSO, prepare 5 mM stock solution</td>
		</tr>
		<tr>
			<td>Starfrost Microscopy Slides</td>
			<td>Waldemar Knittel</td>
			<td>VA11200 003FKB</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen liquid blocker</td>
			<td>Sigma Aldrich</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin funnel</td>
			<td>Q Path Labonord SAS</td>
			<td>003411324</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Red Starter Kit Goat/Rabbit</td>
			<td>Sigma Aldrich</td>
			<td>DUO92105</td>
			<td>Available for different species/combinations, also available in FarRED, Orange and Green</td>
		</tr>
		<tr>
			<td>goat-anti-ATM</td>
			<td>Bethyl Laboratories</td>
			<td>A300-136A</td>
			<td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td>
		</tr>
		<tr>
			<td>rabbit-anti-phospho-Ser15-p53</td>
			<td>Cell Signaling Technology</td>
			<td>9284</td>
			<td>We succesfully used lot #9284-4 in our studies</td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Also available from various other vendors</td>
		</tr>
	</tbody>
</table>

detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay

DNA损伤反应协调自发发生的DNA损伤的修复，由遗传毒性应激引起，或出现在淋巴细胞中程序性DNA断裂的上下文中。共济失调 - Telangiectasia突变激酶（ATM），ATM-和Rad3相关激酶（ATR）和DNA依赖性蛋白激酶（DNA-PKcs）的催化亚基是首先在DNA损伤诱导时被激活，并且是控制DNA修复，凋亡和细胞存活的网络的中央调节剂。作为肿瘤抑制途径的一部分，ATM和ATR通过磷酸化激活p53，从而调节p53的转录活性。 DNA损伤也导致所谓的电离辐射诱导灶（IRIF）的形成，其代表DNA损伤传感器和在DNA损伤部位积聚的修复蛋白质的复合物，其通过荧光显微镜显现。然而，蛋白质在IRIF中的共定位并不一定意味着d直接的蛋白质 - 蛋白质相互作用，因为荧光显微镜的分辨率是有限的。 原位接近连接测定（PLA）是一种新颖的技术，可以以前所未有的特异性和灵敏度直接显示细胞和组织中的蛋白质 - 蛋白质相互作用。该技术基于与感兴趣的蛋白质结合的特异性抗体的空间接近。当询问的蛋白质在〜40nm内时，扩增反应由与抗体缀合的寡核苷酸引发，扩增产物通过荧光标记显现，产生对应于相互作用蛋白质的亚细胞位置的信号。使用ATM和p53之间建立的功能相互作用作为示例，在此证明PLA如何可用于悬浮细胞培养物中以研究作为DNA损伤反应的组成部分的蛋白质之间的直接相互作用。

残留Ser15上p53的磷酸化显示依赖于ATM激酶活性16 。为了证明和证实PLA技术对悬浮细胞培养物细胞分裂素制剂的特异性，表明通过在细胞周期G1期停滞的BCR-ABL + B-ALL细胞2小时NCS处理诱导DNA损伤导致ATM和磷酸化Ser15-p53之间的特异性相互作用，如预期的那样。在用NCS处理的大多数细胞的细胞核中观察到点状PLA信号（ 图1B ），每个细胞平均?...

Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

在这里，证明了原位邻近连接测定（PLA）如何可以用于检测和可视化暴露于基因毒性应激的悬浮细胞培养物中ATM和p53之间的直接蛋白质 - 蛋白质相互作用。

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

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JoVE Journal

Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

利用 tap 标签纯化叶绿体转移蛋白配合物的亲和纯化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...