研究内质网和线粒体相互作用通过<em>原位</em>接近连接分析在固定细胞
Summary
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。
Abstract
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.
Introduction
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>。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。
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。 请点击此处查看该图的放大版本。
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Formaldehyde | Sigma | F-8775 | |
| Glycine | Sigma | G-8898 | |
| Triton | Sigma | T8532 | |
| 35mm Glass bottom culture dishes | MatTeK corporation | P35G-0-14-C | |
| Blocking solution | Sigma | DUO-92004 or DUO-92002 | provided in the Duolink PLA probes, Sigma |
| VDAC1 antibody | Abcam | ab14734 | |
| IP3R1-H80 antibody | Santa Cruz | sc28614 | |
| CypD antibody | Abcam | ab110324 | |
| Grp75 antibody | Santa Cruz | sc13967 | |
| TBS 10X | euromedex | ET220 | Dilute to obtain 1X |
| Tween 100X | euromedex | 2001-B | dilute in TBS to obtain 0,01% |
| PLA Probes Mouse MINUS | Sigma | DUO-92004 | Duolink, Sigma |
| PLA Probes Rabbit PLUS | Sigma | DUO-92002 | Duolink, Sigma |
| Duolink detection reagents red | Sigma | DUO-92008 | Duolink, Sigma |
| Ligation solution | Sigma | DUO-92008 | Part of the Duolink detection reagents red, Sigma |
| Ligase | Sigma | DUO-92008 | Part of the Duolink detection reagents red, Sigma |
| Amplification solution | Sigma | DUO-92008 | Part of the Duolink detection reagents red, Sigma |
| Polymerase | Sigma | DUO-92008 | Part of the Duolink detection reagents red, Sigma |
| Duolink Mounting Medium | Sigma | DUO80102 | Duolink, Sigma |
| Softwares: | |||
| Blob-finder software | 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 at Uppsala University who have developed the software and the work was supported by the EU FP6 Project ENLIGHT and Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm | ||
| ImageJ software | Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html |
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