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

Biz optimize etmek ve protokol doğrulamak katkıda laboratuarımızda tüm insanlara teşekkür ederim. Bu çalışma INSERM ve ulusal araştırma kurumu (ANR-09-JCJC-0116 VE ANR-11-BSV1-033-02) tarafından desteklenmiştir. ET yüksek öğrenim ve araştırma Fransız bakanlığından bir araştırma bursu ile onu doktora sırasında desteklenmiştir.

Çözümler 1. hazırlanması <ol><li> PBS 14.5 ml% 37 formaldehit 5.5 mi seyreltilmesiyle PBS (tuz) içinde% 10 formaldehit hazırlayın. PBS, 50 ml glisin 3.8 g çözülmesiyle, 1 M glisin, pH 8.0, hazırlanması; 1x PBS içinde 100 mM glisin elde etmek için bu seyreltilir. </li><li> 1x PBS içinde% 0.1 Triton-X100 hazırlayın. 3.0 M sodyum klorid ve 25 ° C de pH 7.0 ile, iyonu giderilmiş su içinde hazırlanan 0.30 M trisodyum sitrat ile 20x Tuzlu sodyum sitrat (SSC) hazırlayın. 1x bu tampon sulandırmak ve d...

<ol>
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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...

Mitokondri ve endoplazmik retikulum (ER) hücrede bağımsız organelleri olmayan, ancak mitokondri ilişkili endoplazmik retikulum zarlarının (MAM) olarak tanımlanan temas bölgelerinde yapısal ve fonksiyonel etkileşim. Aslında, MAM her iki taraftan da proteinler arasındaki etkileşimi sağlayan ER membranlar ve mitokondri yakın kapatılan bölgelere karşılık gelir. Bununla birlikte, bu organellerin membranlar bu bölgeler içinde kaynaştırmak yok, bu yüzden onların ayrı varlıklar korumak. MAM Enerji metabolizması ve hücre yaşamını 1-3 etkileyen, mitokondri ER bir kalsiyum açısından önemli bir rol (Ca 2+) ve fosfolipid transferi oynarlar. ER ve mitokondri arasındaki ilişki ilk elektron mikroskobu ile 1970&#39;lerde görüntülendi. O zamandan beri, transmisyon elektron mikroskobu 4,5, elektron tomografi 6,7 veya ER ve mitokondri özgü fluorofor immün lokalizasyonus / floresan proteinleri 8 klasik ER-mitokondri etkileşimleri çalışmak için kullanılmıştır. MAM analizi için yararlı bir araç içi fraksiyonasyon kullanımına dayanır. Bir Percoll degrade 9 bağlanmış diferansiyel Ultrasantrifügasyon tarafından MAM fraksiyonların izolasyonu sağlar. Bununla birlikte, nihai ürün zenginleştirilmiş MAM fraksiyonlar yerine, saf kesimlerin içerir. Toplamda, bu stratejiler özellikle hassas ve / veya kantitatif değildir ve onlar büyük tarama kolayca uygun değildir. Alternatif olarak ilaç indüklenebilir floresan arası organel bağlayıcılar kullanılarak genetik yaklaşımlar ortaya çıkmıştır, ancak protein 10 &#39;nın endojen ekspresyonunun seviyelerinde organel etkileşimlerin analizine izin vermez. MAM 11&#39;de IP3R / GRP75 / SSVD kompleksinin Szabadkai keşfinden dayanarak, ER-mitokondri etkileşimleri analiz etmek için kantitatif bir yöntem geliştirdi. In situ yakınlığı ligati kullanılantahlil üzerinde algılamak ve VDAC1 ve IP3R1 arasındaki etkileşimleri ölçmek için, sabit hücrelerde 12 MAM arayüzünde Ca 2 + -channeling kompleksi yer alan iki organel-yüzey proteinleri. Kısaca, bizler, ER membran dış mitokondriyal zarın (fare anti-VDAC1 birincil antikor) ve IP3R1 (tavşan anti-IP3R1 birincil antikor) (Şekil 1, bir panel) de VDAC1 incelenir. Daha sonra, deney göre, anti-fare ve tamamlayıcı oligonükleotid uzantıları konjuge edilmiş anti-tavşan IgG (fare ve tavşan proksimite ligasyonu deney Probes), her iki ilave edildi. İki hedef proteinler 40 nm&#39;nin altında bir mesafede ise, oligonükleotidler dairesel DNA şablonu (Şekil 1 Panel B) oluşmasına izin vermek için daha sonra ilave bağlayıcı oligo ile hibridize olabilir. Bu dairesel bir DNA molekülü kovalent yakınlığı probları birine bağlanmış bir tek-şeritli DNA ürününü oluşturmak, lige edilmiş ve amplifiye edilir (Şekil 1, Panel C) </stro&gt; Ng. MAM arayüzü ER ve mitokondri arasındaki mesafe mil 6, proksimite ligasyonu ve amplifikasyon bağlı Texas kırmızısı etiketli oligonükleotid sondalar (Şekil 1, Panel D hibridizasyonu sonraki tespiti yol yapılabilir 25-10 nm arasında değerler ). Her floresan nokta böylece tek tek hücrelerin in situ ER-mitokondri etkileşimleri sayılmasına olanak tanıyan, VDAC1 / IP3R1 arasındaki etkileşimleri temsil eder. <img alt="Şekil 1" src="/files/ftp_upload/54899/54899fig1.jpg" /> Şekil 1: Yerinde Proximity ligasyon Tahlili tarafından Endoplazmik Retikulum-mitokondri Etkileşimleri Algılama şematik İllüstrasyon. a) VDAC1 ve MAM arayüzü yakın kendi epitopuna bağlanabilir IP3R1 karşı bir tavşan primer antikora karşı yönlendirilmiş bir fare primer antikor, B) proksimite ligasyonu probları bir çift ilavesifare ve tavşan IgG karşı. Bu sondalar bağlayıcı oligo ligasyonu için şablonlar oluşturabilirler DNA ipliklerini eklenmiş. c) bağlanmasından sonra oluşturulan dairesel DNA zinciri Texas kırmızısı etiketli oligonükleotidler kullanılarak bir flüoresan nokta olarak mikroskopi ile görünür) yükseltilir ve D edilebilir. <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> In situ proksimite ligasyonu deneylerinde benzer antikorların GRP75 / IP3R1 çifti, hem de siklofilin D (CypD) ile yapılabilir / IP3R1 antikorlar CypD MAM arayüzü IP3R / GRP75 / SSVD kompleksi ile etkileştiği gösterilmiştir olduğu dikkate 12-14.

<table border="0" cellpadding="0" cellspacing="0" width="919">
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	</colgroup>
	<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.

Bu protokolü kullanarak bizim deneyime dayalı, biz güvenle sabit hücrelerde ER-mitokondri etkileşimleri görselleştirme ve miktar tayini için bu yöntemi tavsiye ederim. Antikorların çeşitli çiftleri kullanılarak HuH7 hepatokarsinoma hücre çizgisinde in situ proksimite ligasyonu deneyi-görüntülenmiştir ER-mitokondri etkileşimlerine temsili görüntüleri gösterilmektedir. Flüoresans mikroskopisi ile, Şekil 2&#39;de gösterildiği g...

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

Burada, sabit hücrelerde endoplazmik retikulum ve mitokondri arasındaki endojen etkileşimleri görselleştirmek ve yüksek hassasiyet ile ölçmek için bir prosedür açıklanmaktadır. Protokol, bir mitokondri ilişkili zar arayüzünde inositol 1,4,5-trifosfat reseptörü / glukoz-regüle proteini 75 / voltaj bağımlı anyon kanalı / siklofilin D kompleksi hedefleyen in situ yakınlık ligasyonu deneyinde optimize özellikleri.

Endoplazmik retikulum ve mitokondri Etkileşimleri Çalışma ile<em&gt; In Situ</em&gt; Proximity ligasyon Testi Sabit Hücrelerde

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

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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

Efektör-Kompleksleri kullanarak BMP4 Mansabında Sinyal Algılama In situ PLA, Proximity ligasyon Testi

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

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

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

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

Memeli hücrelerinde endoplazmik retikulum lokalize mRNA&#39;ların görselleştirilmesi

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

Gen İfadesi DNA etkileşimleri, Discover Transkripsiyonlu Regülatörleri ve Roman Anti-tümör Ajanlar tanımlayın: Protein Eğitim için Kantitatif Testi

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

MicroRNA<em&gt; In situ</em&gt; Formalin Sabit Böbrek Dokular Hibridizasyon

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

Tek Mitokondriyal süperoksit Bozulmamış Kalp Flaşlar veya konfokal Görüntüleme<em&gt; In Vivo</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

Kültür Hücrelerinin retikulum Subdomain&#39;ler Görselleştirme

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

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

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

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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

Bir kullanılması Endoplazmik retikulum kalsiyum dengesi İzleme<em&gt; Gaussia</em&gt; Lusiferaz SERCaMP

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