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.细胞固定 注意:我们使用在这项研究中的HuH7肝细胞癌细胞系，但这种方法也适用于其他的粘附细胞培养物。 <ol><li>板的HuH7细胞（在DMEM 1g / L的葡萄糖进行培养，补充有10％胎牛血清和0.01％青霉素 - 链霉素的库存）以每未涂覆35毫米玻璃底培养皿150,000个细胞。当原电池工作文化，用胶原蛋白涂层的菜肴。 </li><li>次日，除去培养基。清洗细胞用1毫升PBS和吸出的。通过加入1ml的甲醛10％固定细胞，并孵育在搅拌下室温（RT）10分钟。 </li><li>停止用1ml的1M甘氨酸使反应并通过旋转混合。除去停止反应溶液并通过加入1ml 1×PBS中洗细胞;通过旋转和吸搅动。 1毫升的100mM甘氨酸添加到细胞中，并孵育它们在室温下搅拌15分钟，然后吸出。 注:该协议可以在这里停了下来，下面的步骤可以推迟到另一天。在这种情况下，加入1ml的100mM甘氨酸和，保持在4℃，如果必要的。 </li></ol> 3.细胞透<ol><li>加入1ml 0.1％的Triton-X100的在1×PBS中，在室温下孵育在搅拌下15分钟，然后吸出。上一次电池文化性工作时20分钟 - 在该温育时间可以提高到15ES（ 如初级小鼠肝细胞）。加入1 ml 1X PBS洗细胞;吸出。 </li></ol> 4.封锁<ol><li>添加40微升的封闭溶液以每个样品（由试剂盒提供）;这个体积可以以覆盖样品增加。在湿度室中孵育30分钟的菜在37℃。 </li><li>点击过的菜封闭液。试图获得平等的残液量为每张幻灯片，因为这会影响可重复性。不要让样品干！ </li></ol> 5.一级抗体<ol><li>稀释在1×PBS中的一抗，并添加溶液的培养皿（VDAC1小鼠抗体:1/100，IP3R1兔抗体:1/500）。可替代地，GRP75或CypD抗体（在1/500同时使用小鼠抗体）可以用来代替VDAC1。 </li><li>在4℃的湿度室中孵育过夜。洗载玻片使用的Tris缓冲盐水两次含0.01％吐温（TBS-T）。 </li></ol><p类="jove_title"&gt; 6。接近结扎分析探针<ol><li>接近结扎分析探针由试剂盒。根据初级抗体的物种选择探针。 </li><li>在抗体稀释液5:准备两个接近结扎分析探针1。使混合物静置在室温20分钟。添加稀释接近连接测定探头的解决方案。孵育在预加热的湿度室中的菜肴1小时，在37℃。用TBS-T洗碗两次。 </li></ol> 7.结扎<ol><li>使用在原位检测试剂德克萨斯红套件。 </li><li>稀释5倍结扎股票（由试剂盒提供）1:5的高纯度水拌匀。稀释1倍连接液（由试剂盒提供）连接酶1:40和旋涡。等待添加连接酶，直到立即除了样品之前。 </li><li>添加该溶液至每个样品（40微升为35毫米的玻璃底菜）孵育玻片在预热的湿度湛误码率在37℃，30分钟。用TBS-T洗载玻片两次。 </li></ol> 8.放大注意:小心，光敏感的试剂。 <ol><li>稀释5倍放大股票（由试剂盒提供）1:5的高纯度水。使用冷冻块中删除从冷冻聚合酶（-20℃）。稀释聚合酶（试剂盒提供）1:80在1倍放大的解决方案和旋涡。立即用混合前添加聚合酶。 </li><li>添加该溶液至每个样品（40微升为35毫米的玻璃底菜）。孵育在预加热的湿度室100分钟的幻灯片在37℃。挖掘出幻灯片的扩增聚合酶溶液。 </li></ol> 9.最后经洗涤<ol><li>洗于1×SSC洗涤缓冲液将载玻片2分钟。洗在0.01X SSC洗涤缓冲液将载玻片2分钟。让菜在黑暗中在室温下干燥。 </li></ol> 10.准备成像<ol&gt; <li>使用含有DAPI（水相和不固化）安装介质的最小体积，以确保没有气泡得到盖玻片下抓装入滑动。指甲油可以用来密封边缘。 （:594纳米，发射:624纳米，放大倍数:63X激励）使用荧光或共聚焦显微镜分析前，等待约15分钟。 </li><li>成像后，存放载玻片在黑暗中在-20℃。 </li></ol>

<ol><li>Bravo-Sagua, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organelle+communication%3A+signaling+crossroads+between+homeostasis+and+disease%5BTitle%5D)+AND+%22The+international+journal+of+biochemistry+%26+cell+biology%22%5BJournal%5D)">Organelle communication: signaling crossroads between homeostasis and disease.</a> The international journal of biochemistry & cell biology. 50, 55-59 (2014).</li>
<li>Giorgi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria-associated+membranes%3A+composition%2C+molecular+mechanisms%2C+and+physiopathological+implications%5BTitle%5D)+AND+%22Antioxidants+%26+redox+signaling%22%5BJournal%5D)">Mitochondria-associated membranes: composition, molecular mechanisms, and physiopathological implications.</a> Antioxidants & redox signaling. 22, 995-1019 (2015).</li>
<li>Phillips, M. J., Voeltz, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+function+of+ER+membrane+contact+sites+with+other+organelles%5BTitle%5D)+AND+%22Nature+reviews.+Molecular+cell+biology%22%5BJournal%5D)">Structure and function of ER membrane contact sites with other organelles.</a> Nature reviews. Molecular cell biology. 17, 69-82 (2016).</li>
<li>Cosson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RTM+resistance+to+potyviruses+in+Arabidopsis+thaliana%3A+natural+variation+of+the+RTM+genes+and+evidence+for+the+implication+of+additional+genes%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The RTM resistance to potyviruses in Arabidopsis thaliana: natural variation of the RTM genes and evidence for the implication of additional genes.</a> PLoS One. 7, 39169(2012).</li>
<li>Mannella, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+dynamics+of+the+mitochondrial+inner+membrane+cristae%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Structure and dynamics of the mitochondrial inner membrane cristae.</a> Biochim Biophys Acta. 1763, 542-548 (2006).</li>
<li>Csordas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+features+and+significance+of+the+physical+linkage+between+ER+and+mitochondria%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">Structural and functional features and significance of the physical linkage between ER and mitochondria.</a> The Journal of cell biology. 174, 915-921 (2006).</li>
<li>Mannella, C. A., Buttle, K., Rath, B. K., Marko, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+microscopic+tomography+of+rat-liver+mitochondria+and+their+interaction+with+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Biofactors%22%5BJournal%5D)">Electron microscopic tomography of rat-liver mitochondria and their interaction with the endoplasmic reticulum.</a> Biofactors. 8, 225-228 (1998).</li>
<li>Rizzuto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Close+contacts+with+the+endoplasmic+reticulum+as+determinants+of+mitochondrial+Ca2%2B+responses%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.</a> Science. 280, 1763-1766 (1998).</li>
<li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat Protoc. 4, 1582-1590 (2009).</li>
<li>Csordas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39, 121-132 (2010).</li>
<li>Szabadkai, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chaperone-mediated+coupling+of+endoplasmic+reticulum+and+mitochondrial+Ca2%2B+channels%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels.</a> J Cell Biol. 175, 901-911 (2006).</li>
<li>Tubbs, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria-associated+endoplasmic+reticulum+membrane+%28MAM%29+integrity+is+required+for+insulin+signaling+and+is+implicated+in+hepatic+insulin+resistance%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance.</a> Diabetes. 63, 3279-3294 (2014).</li>
<li>Paillard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depressing+Mitochondria-Reticulum+Interactions+Protects+Cardiomyocytes+From+Lethal+Hypoxia-Reoxygenation+Injury%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Depressing Mitochondria-Reticulum Interactions Protects Cardiomyocytes From Lethal Hypoxia-Reoxygenation Injury.</a> Circulation. 128, 1555-1565 (2013).</li>
<li>Rieusset, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+calcium+transfer+from+ER+to+mitochondria+links+alterations+of+mitochondria-associated+ER+membrane+integrity+to+hepatic+insulin+resistance%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance.</a> Diabetologia. 59, 614-623 (2016).</li>
<li>Allalou, A., Wahlby, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BlobFinder%2C+a+tool+for+fluorescence+microscopy+image+cytometry%5BTitle%5D)+AND+%22Computer+methods+and+programs+in+biomedicine%22%5BJournal%5D)">BlobFinder, a tool for fluorescence microscopy image cytometry.</a> Computer methods and programs in biomedicine. 94, 58-65 (2009).</li>
<li>Theurey, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria-associated+endoplasmic+reticulum+membranes+allow+adaptation+of+mitochondrial+metabolism+to+glucose+availability+in+the+liver%5BTitle%5D)+AND+%22Journal+of+molecular+cell+biology%22%5BJournal%5D)">Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.</a> Journal of molecular cell biology. , (2016).</li>
<li>de Brito, O. M., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitofusin+2+tethers+endoplasmic+reticulum+to+mitochondria%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mitofusin 2 tethers endoplasmic reticulum to mitochondria.</a> Nature. 456, 605-610 (2008).</li>
<li>Soderberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation%5BTitle%5D)+AND+%22Nature+methods%22%5BJournal%5D)">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nature methods. 3, 995-1000 (2006).</li>
<li>De Pinto, V., Messina, A., Lane, D. J., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Voltage-dependent+anion-selective+channel+%28VDAC%29+in+the+plasma+membrane%5BTitle%5D)+AND+%22FEBS+letters%22%5BJournal%5D)">Voltage-dependent anion-selective channel (VDAC) in the plasma membrane.</a> FEBS letters. 584, 1793-1799 (2010).</li>
<li>Kaul, S. C., Taira, K., Pereira-Smith, O. M., Wadhwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mortalin%3A+present+and+prospective%5BTitle%5D)+AND+%22Experimental+gerontology%22%5BJournal%5D)">Mortalin: present and prospective.</a> Experimental gerontology. 37, 1157-1164 (2002).</li>
</ol>

The authors declare that they have no competing financial interests.

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 by immunofluorescence before the proximity ligation assay experiments in order to ensure specificity and to find the correct working dilutions. We also note that the dilution of antibodies needs to be systematically adjusted when using a new proximity ligation assay kit or a new antibody batch. We chose to use in situ detection Texas red reagents, but it is also possible to use reagents in other wavelengths or to use the HRP/Novared kit for bright-field detection. We used this option to analyze ER-mitochondria interactions in paraffin-embedded liver samples16, enabling the visualization of ER-mitochondria interactions as red-brownish dots, thus overcoming the difficulty of working on liver tissue that presents marked autofluorescence.
Compared to conventional imaging approaches6,7,17, the in situ proximity ligation assay18 avoids using organelle-specific fluorophores or fluorescent proteins that could induce some artefacts. Furthermore, it offers improved selectivity because it requires dual recognition and increased sensitivity. It includes DNA amplification as a component for detecting the target protein by using fluorescence microscope. Finally, the procedure is fast, suitable for multiple-condition testing, reproducible, and consistent. Indeed, we systematically found very reproducible results when targeting several pairs of proteins (VDAC/IP3R, GRP75/IP3R, CypD/IP3R) at the MAM interface or when using alternative strategies (electron microscopy analysis, subcellular fractionation, or co-immunoprecipitation)12,13,16, strongly supporting the robustness of the technique and its ability to monitor organelle coupling. Nevertheless, as some targeted proteins (e.g., VDAC and GRP75) have been reported to be present in other intracellular structures19,20, we cannot completely exclude unspecific signals if IP3R is not far away from these structures. However, co-localization studies of each targeted proteins with specific labeling of respective organelles have been performed in HuH7 cells12, and the majority of the signal was merged in each case, suggesting that another localization of proteins, if present, is minor. Therefore, we recommend that this possibility is tested in each investigated cell or tissue and that the proximity ligation assay is performed with the three pairs of antibodies to totally confirm the results. In addition, the expression level of each targeted proteins should be investigated in each case to determine whether the modulation of organelle proximity is linked to a structural modification or to variation in the expression levels of the targeted proteins.
Until now, the lack of adapted analytical methods has prevented dynamic monitoring of ER-mitochondria contact in health and disease. Now, we think that the use of this in situ proximity ligation assay should allow a better understanding of the physiological regulation of ER-mitochondria interactions, as well as of their role in pathological contexts.

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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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><tbody><tr><td>Formaldehyde</td><td>Sigma</td><td>F-8775</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G-8898</td><td></td></tr><tr><td>Triton</td><td>Sigma</td><td>T8532</td><td></td></tr><tr><td>35 mm Glass bottom culture dishes</td><td>MatTeK corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Blocking solution</td><td>Sigma</td><td>DUO-92004 or DUO-92002</td><td>provided in the Duolink PLA probes, Sigma</td></tr><tr><td>VDAC1 antibody</td><td>Abcam</td><td>ab14734</td><td></td></tr><tr><td>IP3R1-H80 antibody</td><td>Santa Cruz</td><td>sc28614</td><td></td></tr><tr><td>CypD antibody</td><td>Abcam</td><td>ab110324</td><td></td></tr><tr><td>Grp75 antibody</td><td>Santa Cruz</td><td>sc13967</td><td></td></tr><tr><td>TBS 10x</td><td>euromedex</td><td>ET220</td><td>Dilute to obtain 1x</td></tr><tr><td>Tween 100x</td><td>euromedex</td><td>2001-B</td><td>dilute in TBS to obtain 0,01%</td></tr><tr><td>PLA Probes Mouse MINUS</td><td>Sigma</td><td>DUO-92004</td><td>Duolink, Sigma</td></tr><tr><td>PLA Probes Rabbit PLUS</td><td>Sigma</td><td>DUO-92002</td><td>Duolink, Sigma</td></tr><tr><td>Duolink detection reagents red</td><td>Sigma</td><td>DUO-92008</td><td>Duolink, Sigma</td></tr><tr><td>Ligation solution</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Ligase</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Amplification solution</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Polymerase</td><td>Sigma</td><td>DUO-92008</td><td>Part of the Duolink detection reagents red, Sigma</td></tr><tr><td>Duolink Mounting Medium</td><td>Sigma</td><td>DUO80102</td><td>Duolink, Sigma</td></tr><tr><td colspan="4">&lt;strong&gt;Softwares&lt;/strong&gt;</td></tr><tr><td>Blob-finder software</td><td></td><td></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 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</td></tr><tr><td>ImageJ software</td><td></td><td></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，只使用一个一级抗体导致了无信号（图2，图a），验证双重识别要求用于原位接近连接测定信号产生。经典地，我们分析使用VDAC1 / IP3R1对抗体（图2，图b）的ER-线粒体相互作用。 原位接近结扎法experimen类似TS也可以与GRP75 / IP3R1或CypD / IP3R1对抗体（ 图2，板c和d，分别）的执行中，由于GRP75是分子伴侣在MAM接口11耦合VDAC和IP3受体和CypD被证明与交互该VDAC / GRP75 / IP3R 钙 -channeling复杂12-14。可以使用任何的Blob取景器15或ImageJ的软件进行荧光点组成的量化。作为细胞的细胞核中的蓝色标记，每个小区对ER-线粒体相互作用quantifications是适于执行。我们彻底地验证了该法12，我们可以得出结论，这些有针对性的蛋白质是很好的候选人，研究ER-线粒体相互作用。 <img alt="图2" src="/files/ftp_upload/54899/54899fig2.jpg" /> 图2:VDAC1 / IP3R1，GRP75 / IP3R1和CypD / IP3R1互动的可视化的原位接近Ligatioñ分析在Huh7细胞。细胞核显示为蓝色，并且两个目标蛋白质之间的相互作用在红色被描绘（63X放大倍率;比例尺= 20微米）。我们表明，VDAC1，GRP75，和CypD与IP3R1交互。作为阴性对照，我们表明，只有一个初级抗体不会导致红色荧光。为了验证分析中，其它几个控制已经进行了证明VDAC1，GRP75，和CypD不与其他的ER蛋白质相互作用和IP3R1不与其他线粒体蛋白质相互作用（数据未示出，请参阅参考文献9和13）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54899/54899fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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), ...

study-endoplasmic-reticulum-mitochondria-interactions-situ-proximity

Nanyang Technological University

Research

JoVE Journal

Biology

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

24.6K 次观看.  Lund University. 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。 

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

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

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

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

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

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

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

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

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

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

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 ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

发育生物学

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

神经科学

<meta charset="utf8"></head><body>一种用于高通量筛选的测量 ER-线粒体接触的简单检测

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>作者聚焦:ER-线粒体接触的调节和失调 — 对神经退行性疾病发病机制的影响

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

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 ...

生物学

Correlative Microscopy Reveals Stress-Induced Mitochondria-Lysosome Interactions

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for ...

研究内质网和线粒体相互作用通过

摘要

探索更多视频

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和

原位近接结扎法检测蛋白异构体的 Heterodimerization

线粒体和内质视网膜成像由相关光和体积电子显微镜

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

检测

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

裂解荧光素酶重组测定法测量活细胞中内质网-线粒体接触

内质网子域的培养细胞中的可视化

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes