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

Wij danken alle mensen in ons laboratorium die hebben bijgedragen aan het optimaliseren en valideren van het protocol. Dit werk werd ondersteund door INSERM en de nationale onderzoeksbureau (ANR-09-JCJC-0116 EN ANR-11-BSV1-033-02). ET werd gesteund tijdens haar promotieonderzoek door een onderzoeksbeurs van het Franse ministerie van hoger onderwijs en onderzoek.

1. Voorbereiding van de Solutions <ol><li> Bereid 10% formaldehyde in PBS (laag zout) door verdunning van 5,5 ml 37% formaldehyde in 14,5 ml PBS. Bereid 1 M glycine, pH 8,0, door het oplossen van 3,8 g glycine in 50 ml PBS; Verdun deze oplossing 100 mM glycine verkrijgen 1x PBS. </li><li> Bereid 0,1% Triton-X100 in 1x PBS. Bereid 20x Saline natriumcitraat (SSC) met 3,0 M natriumchloride en 0,30 M trinatriumcitraat bereid in gedeïoniseerd water buffer met pH 7,0 bij 25 ° C. Verdun deze buffer om 1x en 0.01x met behul...

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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...

Mitochondria en het endoplasmatisch reticulum (ER) zijn niet onafhankelijk organellen in de cel, maar ze omgaan structureel en functioneel in contact plaatsen gedefinieerd als mitochondriën verbonden endoplasmatisch reticulum membranen (MAM). In feite, MTM overeen met gebieden waar de membranen van het ER en mitochondria nauw apposed, zodat interacties tussen eiwitten van beide kanten. Toch weet de membranen van deze organellen niet fuseren binnen deze regio&#39;s, zodat ze behouden hun afzonderlijke entiteiten. De MTM spelen een cruciale rol in de calcium (Ca 2+) en fosfolipiden transfer van ER naar mitochondriën, invloed energiemetabolisme en celoverleving 1-3. De associatie tussen het ER en mitochondria werd eerst gevisualiseerd in de jaren 1970 met elektronenmicroscopie. Sindsdien, transmissie-elektronenmicroscopie 4,5, electron tomography 6,7 of immuno-lokalisatie van ER en mitochondriën-specifieke fluorofoors / fluorescente proteïnen werden 8 klassiek gebruikt om ER-mitochondriën interacties te bestuderen. Een ander bruikbaar middel voor de analyse van MAM is gebaseerd op het gebruik van subcellulaire fractionering. Het laat de isolatie van MAM fracties door differentiële ultracentrifugatie gekoppeld met een Percoll gradiënt 9. De eindproduct bevat MAM verrijkte fracties, dan zuivere fracties. In totaal hebben deze strategieën niet bijzonder gevoelig en / of kwantitatieve en zijn lastig om grote screening. Alternatief genetische benadering en de geneesmiddel-geïnduceerd fluorescent inter-organel linkers zijn ontstaan, maar niet de analyse van organel interacties zodat het endogene expressieniveaus van eiwitten 10. Op basis van de ontdekking Szabadkai&#39;s van de IP3R / GRP75 / VDAC complex in het MAM 11, ontwikkelden we een kwantitatieve methode om de ER-mitochondria interacties te analyseren. We gebruikten de in situ omgeving ligatiOp assay voor het detecteren en kwantificeren van interacties tussen VDAC1 en IP3R1 twee organel-oppervlakte-eiwitten betrokken zijn bij de Ca2 + -channeling complex in het MAM interface gefixeerde cellen 12. Samengevat, zijn we gehybridiseerd VDAC1 het buitenste mitochondriale membraan (muis anti-VDAC1 primair antilichaam) en IP3R1 het ER membraan (konijn anti-IP3R1 primaire antilichaam) (Figuur 1, panel a). Vervolgens wordt aan de test, voegden we zowel anti-muis en anti-konijn IgG (muis en konijn nabijheid ligatie assay probes) die zijn geconjugeerd met complementaire oligonucleotide extensies. Als de twee eiwitten gericht zijn op een afstand onder 40 nm, kunnen de oligonucleotiden hybridiseren met de later toegevoegde connector oligo om de vorming van een circulair DNA-matrijs (Figuur 1, paneel b) toestaan. Deze circulaire DNA molecuul wordt geligeerd en geamplificeerd, waardoor een enkelstrengs DNA product covalent aan één van de probes nabijheid (Figuur 1, panel c) </strong&gt;. Aangezien de afstand tussen het ER en mitochondriën bij de MAM-interface varieert van 10 nm tot 25 nm 6, nabijheid ligatie en amplificatie kan leiden tot daaropvolgende detectie door hybridisatie van Texas rood gemerkte oligonucleotiden probes (Figuur 1, panel d ). Elke fluorescerende stip staat wisselwerkingen tussen VDAC1 / IP3R1, waardoor de kwantificering van in situ ER-mitochondriën interacties in individuele cellen. <img alt="Figuur 1" src="/files/ftp_upload/54899/54899fig1.jpg" /> Figuur 1: Schematische weergave van de Opsporing van het endoplasmatisch reticulum-mitochondria interacties van In Situ Proximity Ligatie Assay. a) Een muis primair antilichaam gericht tegen VDAC1 en een konijn primair antilichaam tegen IP3R1 kan binden aan hun epitopen in de nabijheid van het MAM-interface, b) de toevoeging van een paar proximity ligatieprobesgericht tegen muizen en konijnen IgG. Deze sondes DNA strengen die sjablonen voor de ligatie van oligonucleotiden connector kan vormen verbonden. c) De circulaire DNA-streng gevormd na ligatie kunnen worden geamplificeerd en d) gevisualiseerd door microscopie als fluorescerend dot met Texas-rood gemerkte oligonucleotiden. <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">Klik hier om een grotere versie van deze figuur te bekijken.</a> Vertaald in situ nabijheid ligatie assay experimenten kunnen worden uitgevoerd met de GRP75 / IP3R1 paar antilichamen, evenals cyclofiline D (CypD) / IP3R1 antilichamen, aangezien CypD bleek interactie met de IP3R / GRP75 / VDAC complex in het MAM-interface 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.

Op basis van onze ervaring met dit protocol, kunnen we gerust deze methode voor de visualisatie en kwantificatie van ER-mitochondria interacties in vaste cellen aanraden. Representatieve beelden van in situ nabijheid ligation-assay gevisualiseerd ER-mitochondria interacties in de HuH7 hepatocarcinoma cellijn, met behulp van verschillende paren van antilichamen, worden getoond. Zoals getoond in figuur 2 door fluorescentiemicroscopie, elke rode stip voor een wisse...

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

Hier beschrijven we een werkwijze voor het visualiseren en kwantificeren gevoeligheid een endogene interacties tussen het endoplasmatisch reticulum en mitochondria in gefixeerde cellen. Het protocol is voorzien van een geoptimaliseerde in situ nabijheid ligation assay gericht op de inositol 1,4,5-trifosfaat receptor / glucose-gereguleerd eiwit 75 / voltage-afhankelijke anion kanaal / cyclofiline D-complex aan de mitochondria bijbehorende membraan interface.

Studie van endoplasmatisch reticulum en mitochondriën interacties door<em&gt; In Situ</em&gt; Proximity Ligatie Assay in gefixeerde cellen

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

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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

Een Kwantitatieve Analyse naar Protein Studie: DNA interacties, Discover transcriptieregulatoren van genexpressie, en het identificeren van nieuwe anti-tumor Agents

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; Hybridisatie voor formaline gefixeerde nierweefsel

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

Confocale beeldvorming van Single mitochondriaal superoxide Knippert Intact Heart of<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

Visualisatie van endoplasmatisch reticulum subdomeinen bij gekweekte cellen

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

Profilering Individuele menselijke embryonale stamcellen door kwantitatieve 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

In Vivo 4-dimensionale Tracking van hematopoietische stamcellen en progenitorcellen in volwassen muis calvarial Bone Marrow

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

Monitoring endoplasmatisch reticulum calciumhomeostase Met behulp van een<em&gt; Gaussia</em&gt; Luciferase SERCaMP

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