Name: study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells
Uploaded: 2016-12-10T14:00:00
Description: Watch this Scientific Journal Video about Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells at JoVE.com

我们感谢所有在我们的实验室谁促成了优化和验证协议的人。这项工作是由INSERM和国家研究机构（ANR-09-JCJC-0116和ANR-11-BSV1-033-02）的支持。 ET博士学位由高等教育和研究的法国文化部的一个研究奖学金期间的支持。

1.溶液的制备<ol><li>通过稀释5.5毫升37％甲醛的14.5毫升的PBS制备于PBS（低盐）10％的甲醛。制备1M甘氨酸，pH 8.0的，溶解在50ml的PBS将3.8g甘氨酸;稀释此溶液，得到在1×PBS中的100mM甘氨酸。 </li><li>制备0.1％的Triton-X100在1×PBS中。通过使用3.0M的氯化钠和0.30柠檬酸三钠在25℃在去离子水缓冲液中制备具有pH为7.0制备20倍盐水柠檬酸钠（SSC）。这稀释缓冲1X和使用去离子水0.01X。 </li></ol> 2.细...

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Collectively, our studies indicate that the in situ proximity ligation assay is truly a relevant strategy to follow and quantify endogenous ER-mitochondria interactions in fixed cells, without the need for using organelle-specific fluorophores or fluorescent proteins. The specific use of VDAC1/IP3R1 antibodies has been adapted to study ER-mitochondria interactions in HuH7 cells. However, alternative isoforms of VDAC and IP3R may be used, depending on the cell type. In this case, antibodies need to be validated b...

线粒体和内质网（ER）是不是在细胞中独立的细胞器，但它们在结构上和功能上相互作用在定义为线粒体相关的内质网的膜（MAM）的接触位点。事实上，MAMS对应其中ER的膜和线粒体紧密并列的区域，使从两侧的蛋白质之间的相互作用。然而，这些细胞器的膜不这些区域内熔合，因此它们保持它们的独立的实体。该MAMS起到钙（Ca 2+）关键作用，磷脂转移从ER到线粒体，影响能量代谢和细胞存活1-3。 内质网和线粒体之间的关系在20世纪70年代用电子显微镜首次观察。自那时以来，透射电子显微镜4,5，电子断层扫描6,7或ER和特定线粒体-荧光团的免疫定位S /荧光蛋白8人经典用于研究ER-线粒体相互作用。为MAM的分析的另一个有用的工具是基于使用亚细胞分离的。它允许MAM馏分的隔离通过耦合到Percoll密度梯度9差动超速离心。然而，最终产物含有丰富的MAM馏分，而不是纯的级分。总之，这些策略没有特别敏感和/或定量的，它们是不容易适合于大的筛选。可替代地，使用药物诱导的荧光间细胞器的接头遗传方法已经出现，但它们不允许细胞器相互作用的分析在蛋白10的内源表达水平。 根据Szabadkai的发现的IP3受体/ GRP75 / VDAC络合物在MAM 11中 ，我们开发了一个定量的方法来分析ER-线粒体相互作用。我们使用原位接近ligati上测定来检测和量化VDAC1和IP3R1之间的相互作用，在固定单元12参与在MAM接口的钙 -channeling复杂二细胞器表面蛋白。简言之，我们在线粒体外膜（小鼠抗VDAC1初级抗体）与IP3R1在ER膜（兔抗IP3R1初级抗体）（图1，分图a）探测VDAC1。然后，根据该测定，我们增加既抗小鼠和抗兔IgG（小鼠和兔接近结扎分析探针），其偶联至互补寡核苷酸的扩展。如果两个目标蛋白质是在低于40纳米的距离，寡核苷酸可以与随后加入的接头的寡核苷酸杂交以允许环状DNA模板（图1，图b）的形成。此环形DNA分子连接并放大，产生共价连接到邻近探针之一的单链DNA产物（图1，分图c） </stroNG&gt;。由于在MAM接口的ER和线粒体之间的距离为10纳米的范围内，以25纳米6，接近结扎和扩增可以完成，从而导致后续检测由于德克萨斯红标记的寡核苷酸探针（图1，图d的杂交）。每个荧光点代表VDAC1 / IP3R1之间的相互作用，因而允许在单个细胞中原位 ER-线粒体相互作用的定量。 <img alt="图1" src="/files/ftp_upload/54899/54899fig1.jpg" /> 图1：由内质网线粒体相互作用检测的示意图原位接近结扎分析。 一）针对VDAC1和针对IP3R1可以在MAM接口绑定到其表位接近兔第一抗体的小鼠初级抗体， 二）除了一对接近结扎探针的针对小鼠和兔IgG。这些探针已附加的DNA链，可以形成用于连接寡核苷酸的结扎模板。 三）结扎后所形成的环状DNA链可以通过使用德克萨斯红标记的寡核苷酸扩增和d）通过显微镜观察作为荧光点。 <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 原位接近连接测定实验相似可以与GRP75 / IP3R1对抗体，以及亲环D（CypD）来执行/ IP3R1抗体，考虑到CypD被证明与在MAM接口的IP3受体/ GRP75 / VDAC复合物相互作用12-14。

<table border="0" cellpadding="0" cellspacing="0" width="919">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Formaldehyde</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">F-8775</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Glycine</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">G-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Triton</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">35mm Glass bottom culture dishes</td>
			<td style="width:169px;">MatTeK corporation</td>
			<td style="width:187px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blocking solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004 or DUO-92002</td>
			<td>provided in the Duolink PLA probes, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">VDAC1 antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab14734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IP3R1-H80 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc28614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CypD antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab110324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Grp75 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc13967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">TBS 10X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">ET220</td>
			<td>Dilute to obtain 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tween 100X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">2001-B</td>
			<td>dilute in TBS to obtain 0,01%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Mouse MINUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Rabbit PLUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92002</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink detection reagents red</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligation solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Amplification solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Polymerase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink Mounting Medium</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO80102</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Softwares:</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blob-finder software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>BlobFinder is a freely distributed software that can perform calculations on cells from fluorescence microscopy images. This software can be downloaded for free from The Centre for Image Analysis&#160;at Uppsala University who have developed the software and the work was supported by the EU FP6 Project&#160;ENLIGHT&#160;and&#160;&#160;Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">ImageJ software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html</td>
		</tr>
	</tbody>
</table>

study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

基于使用此协议我们的经验，我们可以放心地推荐为固定细胞ER-线粒体相互作用的可视化和量化这种方法。的在的HuH7肝癌细胞原位接近连接测定-可视ER-线粒体的相互作用，用几对抗体的代表性图像，被示出。如在图2中由荧光显微镜所示，每个红点代表两个目标蛋白质之间的相互作用，以及细胞核显示为蓝色。作为阴性对照，只使用一个一级抗?...

Watch this Scientific Journal Video about Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells at JoVE.com

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

这里，我们描述一个过程中固定的细胞可视化和高灵敏度量化内质网和线粒体之间的内源的相互作用。该协议提供在原位接近连接测定在线粒体相关膜界面靶向1,4,5-三磷酸肌醇受体/葡萄糖调节蛋白75 /电压依赖性阴离子通道/亲环三维复杂的优化。

研究内质网和线粒体相互作用通过<em&gt;原位</em&gt;接近连接分析在固定细胞

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Research

JoVE Journal

Biology

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

检测信号效应复合物BMP4的下游使用原位解放军，接近结扎分析

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

科研

生物学

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...