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

Vi takker alle menneskene i vårt laboratorium som har bidratt til å optimalisere og validere protokollen. Dette arbeidet ble støttet av INSERM og nasjonale forskningsbyrået (ANR-09-JCJC-0116 OG ANR-11-BSV1-033-02). ET ble støttet i løpet av sin doktorgrad ved en stipendiatstilling fra det franske departementet for høyere utdanning og forskning.

1. Utarbeidelse av Solutions <ol><li> Fremstille 10% formaldehyd i PBS (lav-salt) ved å fortynne 5,5 ml 37% formaldehyd i 14,5 ml PBS. Fremstille en M glycin, pH 8,0, ved oppløsning av 3,8 g glycin i 50 ml PBS; fortynne denne løsningen for å oppnå 100 mM glycin i 1 x PBS. </li><li> Forbered 0,1% Triton-X100 i 1x PBS. Forbered 20x Saline natriumcitrat (SSC) ved bruk av 3,0 M natriumklorid og 0,30 M trinatriumcitrat fremstilt i et avionisert vann buffer med pH 7,0 ved 25 ° C. Fortynn denne bufferen til 1x og 0.01x...

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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...

Mitokondrier og endoplasmatisk retikulum (ER) er ikke uavhengige organeller i cellen, men de interagerer strukturelt og funksjonelt at kontaktsider er definert som mitokondrier-assosiert endoplasmatiske retikulum membraner (MAM). Faktisk, MAMS tilsvarer regioner hvor membranene ifølge ER og mitokondrier er tett av motstå, slik at interaksjonene mellom proteiner fra begge sider. Likevel, gjør membraner av disse organeller ikke smelter innenfor disse områdene, slik at de opprettholder sine separate enheter. De Mams spiller en avgjørende rolle i kalsium (Ca 2+) og fosfolipid overføring fra ER til mitokondriene, påvirker energiomsetningen og celle overlevelse 1-3. Sammenhengen mellom ER og mitokondriene ble først visualisert i 1970 med elektronmikroskopi. Siden da transmisjonselektronmikroskopi 4,5, elektron tomografi 6,7 eller immuno-lokalisering av ER og mitokondrier-spesifikke fluoroforens / fluorescerende proteiner 8 ble klassisk brukt til å studere ER-mitokondrier interaksjoner. En annen nyttig verktøy for analyse av MAM er basert på bruk av subcellulære fraksjonering. Det tillater isolering av MAM-fraksjoner ved differensial-ultrasentrifugering koblet til en Percoll-gradient 9. Imidlertid inneholder det endelige produktet anrikede MAM fraksjoner, snarere enn rene fraksjoner. Til sammen er disse strategiene er ikke spesielt følsomme og / eller kvantitativ, og de er ikke lett mottagelig for stor screening. Alternativt har genetiske metoder ved hjelp av narkotika induserbar fluorescerende inter-organelle linkere dukket opp, men de tillater ikke analysen av organelle interaksjoner på endogene uttrykk nivåer av proteiner 10. Basert på Szabadkai oppdagelse av IP3R / GRP75 / VDAC kompleks på MAM 11, har vi utviklet en kvantitativ metode for å analysere ER-mitokondrier interaksjoner. Vi brukte in situ nærhet ligatipå analyse for å påvise og kvantifisere interaksjoner mellom VDAC1 og IP3R1, to organelle-overflateproteiner involvert i Ca 2+ -channeling komplekse på MAM-grensesnittet i faste celler 12. I korthet, probet vi VDAC1 ved den ytre mitokondriemembranen (mus anti-VDAC1 primære antistoff) og IP3R1 ved ER-membranen (kanin-anti-IP3R1 primære antistoff) (figur 1, felt A). Deretter, i henhold til analysen, tilsatte vi både anti-mus og anti-kanin-IgG (mus og kanin nærhet ligering assay-prober), som er konjugert til komplementære oligonukleotid utvidelser. Hvis de to målrettede proteiner er i en avstand under 40 nm, kan oligonukleotidene hybridisere med de etterfølgende ekstra koblings oligoer for å tillate dannelsen av et sirkulært DNA-templat (figur 1, felt B). Dette sirkulært DNA-molekyl blir ligert og forsterkes, noe som skaper et enkelt-trådet DNA-produkt kovalent bundet til en av nærhets prober (figur 1, felt C) </strong&gt;. Da avstanden mellom ER og mitokondrier på MAM-grensesnittet i området fra 10 nm til 25 nm 6, kan nærhet ligering og forsterkning gjøres, noe som fører til etterfølgende påvisning på grunn av hybridisering av Texas Rødt-merkete oligonukleotider sonder (figur 1, panel d ). Hver fluorescerende prikk representerer interaksjoner mellom VDAC1 / IP3R1, og dermed gir kvantifisering av in situ ER-mitokondrier interaksjoner i enkeltceller. <img alt="Figur 1" src="/files/ftp_upload/54899/54899fig1.jpg" /> Figur 1: Skjematisk illustrasjon av Gjenkjenning av det endoplasmatiske retikulum-mitokondrier Interaksjoner med In Situ Proximity hemorroider analysen. a) En mus primært antistoff rettet mot VDAC1 og et kanin primært antistoff rettet mot IP3R1 kan binde seg til sine epitoper i nærhet ved MAM grensesnitt, b) Tilsetning av et par av nærhetsligerings proberrettet mot mus og kanin IgG. Disse probene har festet DNA-strengene som kan danne maler for ligering av koblings oligos. c) Den sirkulære DNA-tråd dannet etter ligering kan forsterkes og d) visualiseres ved mikroskopi som et fluorescerende punkt ved hjelp av Texas Rødt-merkete oligonukleotider. <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a> Ligner in situ nærhet Ringsanalyse eksperimenter kan utføres med GRP75 / IP3R1 par av antistoffer, samt cyklofilin D (CypD) / IP3R1 antistoffer, med tanke på at CypD ble vist å samhandle med IP3R / GRP75 / VDAC komplekse på MAM-grensesnitt 12-14.

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

Basert på vår erfaring med denne protokollen, kan vi trygt anbefale denne metoden for visualisering og kvantifisering av ER-mitokondrier interaksjoner i faste celler. Representative bilder av in situ nærhet Ringsanalyse-visualisert ER-mitokondrier interaksjoner i HuH7 hepatokarsinom cellelinje, ved hjelp av flere par av antistoffer, blir vist. Som vist i figur 2 av fluorescerende mikroskopi, representerer hver rød prikk en interaksjon mellom de to målretted...

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

Her beskriver vi en prosedyre for å visualisere og kvantifisere med høy følsomhet endogene samspillet mellom det endoplasmatiske retikulum og mitokondrier i faste celler. Protokollen har en optimalisert in situ nærhet ligation analysen rettet mot inositol 1,4,5-trifosfat reseptor / glukose-regulert protein 75 / spenningsavhengig anion kanal / cyklofilin D komplekse på mitokondriene assosierte membran grensesnitt.

Studier av endoplasmatiske retikulum og mitokondrier Interaksjoner med<em&gt; In Situ</em&gt; Nærhet hemorroider analysen i fikserte celler

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

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Biology

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

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

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Gjør-det-selv Enhet for gjenoppretting av Cryopreserved Prøver uhell falt inn Kryogeniske lagertanker

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

Visualisering av endoplasmatisk retikulum Lokalisert mRNAs i pattedyrceller

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

En kvantitativ analyse for å studere Protein: DNA interaksjoner, Discover Transcriptional regulatorer Gene Expression, og identifisere nye anti-tumor Agenter

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

MikroRNA<em&gt; In situ</em&gt; Hybridisering for formalinfiksert Nyre Vev

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

Confocal Imaging av enkelt Mitokondrielle superoksid Blinker Intakt hjerte eller<em&gt; I 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

Visualisering av endoplasmatiske retikulum underdomener i dyrkede celler

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 Individuell humane embryonale stamceller ved kvantitativ 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-Dimensional Sporing av Hematopoietiske Stem og stamceller i Adult Mouse 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

Overvåking endoplasmic nettet kalsiumhomeostase Ved hjelp av en<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 ...