Name: sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima
Uploaded: 2018-08-12T14:00:00
Description: Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Dette arbejde blev støttet af en bevilling fra National Research Foundation i Korea (2016R1D1A1B03931949) (til J. U.) og af Grundforskningsfond af Korea (NRF) tilskud finansieret af Korea government(MSIT) (nr. 2016R1A2B2008887, nr. 2016R1A5A2007009) (til J.Y .)

1. generation af HeLa celler udtrykker mito-Keima (mt-Keima)
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Forberedelse af mt-Keima lentivirus
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<li>Coat en 100-mm kultur parabol ved tilsætning 2 mL af 0,001% poly-L-lysin/fosfatbufferet saltopløsning (PBS) og lad det stå i 5 min ved stuetemperatur.</li>
		<li>Fjerne den poly-L-lysin løsning ved hjælp af et glas pipette tilsluttet en vacuum og vaske kultur parabol ved tilsætning 2 mL 1 x PBS.</li>
		<li>Plade 1,5 x 106 HEK293T celler på den coatede kultur fad med 10 m...

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+pink1+is+required+for+mitochondrial+function+and+interacts+genetically+with+parkin.">Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.</a> Nature. 441 (7097), 1162-1166 (2006).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+Drosophila+PINK1+mutants+is+complemented+by+parkin.">Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin.</a> Nature. 441 (7097), 1157-1161 (2006).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SESN2/sestrin2+suppresses+sepsis+by+inducing+mitophagy+and+inhibiting+NLRP3+activation+in+macrophages.">SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages.</a> Autophagy. 12 (8), 1272-1291 (2016).</li><li>Kim, M. J., Yoon, J. H., Ryu, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+A+balance+regulator+of+NLRP3+inflammasome+activation.">Mitophagy: A balance regulator of NLRP3 inflammasome activation.</a> BMB Reports. 49 (10), 529-535 (2016).</li><li>Sandoval, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+Nix+in+autophagic+maturation+of+erythroid+cells.">Essential role for Nix in autophagic maturation of erythroid cells.</a> Nature. 454 (7201), 232-235 (2008).</li><li>Sato, M., Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+paternal+mitochondria+by+fertilization-triggered+autophagy+in+C.+elegans+embryos.">Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos.</a> Science. 334 (6059), 1141-1144 (2011).</li><li>Geisler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1/Parkin-mediated+mitophagy+is+dependent+on+VDAC1+and+p62/SQSTM1.">PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.</a> Nature Cell Biology. 12 (2), 119-131 (2010).</li><li>Matsuda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1+stabilized+by+mitochondrial+depolarization+recruits+Parkin+to+damaged+mitochondria+and+activates+latent+Parkin+for+mitophagy.">PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.</a> The Journal of Cell Biology. 189 (2), 211-221 (2010).</li><li>Allen, G. F., Toth, R., James, J., Ganley, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+iron+triggers+PINK1/Parkin-independent+mitophagy.">Loss of iron triggers PINK1/Parkin-independent mitophagy.</a> EMBO Reports. 14 (12), 1127-1135 (2013).</li><li>Chu, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+externalization+to+the+outer+mitochondrial+membrane+acts+as+an+elimination+signal+for+mitophagy+in+neuronal+cells.">Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.</a> Nature Cell Biology. 15 (10), 1197-1205 (2013).</li><li>Kubli, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1+Is+dispensable+for+mitochondrial+recruitment+of+Parkin+and+activation+of+mitophagy+in+cardiac+myocytes.">PINK1 Is dispensable for mitochondrial recruitment of Parkin and activation of mitophagy in cardiac myocytes.</a> PloS One. 10 (6), e0130707 (2015).</li><li>Dolman, N. J., Chambers, K. M., Mandavilli, B., Batchelor, R. H., Janes, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+techniques+to+measure+mitophagy+using+fluorescence+microscopy.">Tools and techniques to measure mitophagy using fluorescence microscopy.</a> Autophagy. 9 (11), 1653-1662 (2013).</li><li>Kuma, A., Matsui, M., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+an+autophagosome+marker,+can+be+incorporated+into+protein+aggregates+independent+of+autophagy:+caution+in+the+interpretation+of+LC3+localization.">LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization.</a> Autophagy. 3 (4), 323-328 (2007).</li><li>Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+quantitative+technique+for+detecting+autophagic+events+based+on+lysosomal+delivery.">A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.</a> Chemistry &amp; Biology. 18 (8), 1042-1052 (2011).</li><li>Lee, I. 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Vi præsenterer her, en hurtig og følsom metode for at bruge flowcytometri at måle cellulære mitophagy aktivitet i celler, der udtrykker mt-Keima. Celler under en høj grad af mitophagy udviser en øget ratio af PE-CF594 (561 nm) / BV605 (405 nm) excitation. Mitophagy aktivitet kan således udtrykkes som procentdelen af cellerne, udstiller høj 561/405 forholdet. Vi beregnet procentdelen af celler i regionen "høj" gate på en prik plot af PE-CF594 (561 nm) versus BV605 (405 nm), og resultaterne viste, at behandling m...

Mitokondrier er organelles, der er nødvendige for celledelingen og fysiologi. Mitokondrier er ansvarlig for at generere mere end 80% af ATP levering via oxidativ fosforylering, og de giver også forskellige metaboliske mellemprodukter til biosyntese og metabolisme1,2. Ud over deres rolle i energiforsyningen og metabolisme spille mitokondrier centrale roller i mange andre vigtige processer, herunder reaktive ilt arter (ROS) generation, regulering af celledød og intracellulære Ca2 + dynamics3 . Ændringer i mitokondrie-funktion er blevet forbundet med menneskelige sygdomme, herunder kræft, diabetes mellitus og forskellige neurodegenerative sygdomme4,5. Øget mitokondriel dysfunktion og mitokondriel DNA mutationer er også menes at bidrage til normal aldring processer6,7,8. Kvalitetskontrol er derfor et afgørende spørgsmål i mitokondrierne. Mitokondrier er yderst dynamisk organelles, der løbende kan ændre deres form mellem aflange netværk og korte, fragmenterede form. Mitokondrier dynamics spiller en vigtig rolle i at bevare funktionen af mitokondrier samt deres nedbrydning gennem mitophagy9.
Mitophagy er en mekanisme, der involverer den selektive nedbrydning af hele mitokondrier ved hjælp af autophagy maskiner. Mitophagy er principielt mekanisme underliggende mitokondrie omsætning og fjernelse af beskadigede eller dysfunktionelle mitochondrier. I denne proces, er mitokondrier først omgivet af en membran, som resulterer i dannelsen af autophagosomes, som derefter fusionere med lysosomer for hydrolytisk nedbrydning9. Tidligere genetiske undersøgelser i Drosophila har antydet, at to Parkinsons sygdom-relaterede gener, PT-induceret formodede kinase 1 (PINK1) (PARK6) og Parkin (PARK2), fungerer i den samme vej10,11. Delfølge undersøgelser har vist at PINK1-Parkin pathway er ansvarlig for at fjerne beskadigede mitokondrier og mangler i denne pathway resulterer i dårligt fungerende mitophagy og kan bidrage til menneskelige sygdomme12,13. Defekter i mitophagy processer er for nylig blevet fundet i forskellige menneskelige sygdomme, herunder kræft, hjertesygdomme, leversygdomme og neurodegenerative sygdomme 14. Derudover har nylige undersøgelser også vist, at mitophagy er afgørende for mange fysiologiske processer, som differentiering, udvikling og immunrespons15,16,17,18 ,19, tyder på, at mitophagy kan spille en mere aktiv rolle i at kontrollere cellefunktioner.
Trods de seneste bekræftelse af, at mitophagy spiller en vigtig rolle i begge kvalitetskontrol i mitokondrierne og sygdom hos mennesker, de molekylære mekanismer bag mitophagy forbliver dårligt forstået. Selv om mitophagy-relaterede mekanismer er blevet systematisk undersøgt i gær, har studier med henblik på at udforske mitophagy-relaterede mekanismer i pattedyrceller primært fokuseret på PINK1-Parkin pathway. Tidligere undersøgelser har godtgjort, at PINK1-Parkin pathway er hovedansvarlig for den selektive fjernelse af beskadiget mitokondrier via mitophagy12,20,21. Men de seneste undersøgelser har rapporteret, at i nogle modeller kan aktiveres mitophagy selv i mangel af funktionelle PINK122,23,24. Disse resultater tyder på, ud over PINK1-Parkin pathway, der en yderligere uidentificerede pathway, der kan aktivere mitophagy.
Fraværet af en praktisk metode til at vurdere mitophagy aktivitet er en væsentlig hindring for at udforske de stier, der regulerer mitophagy og dens rolle i patofysiologiske begivenheder. Elektronmikroskopi er et kraftfuldt værktøj til direkte visualisere autophagosome-opslugte mitokondrier. Elektronmikroskopi har imidlertid begrænsninger i kvantificere mitophagy aktivitet. Selv om strategier, der bruger mikrotubulus-forbundet protein-1 lys kæde 3 (LC3)-konjugeret fluorescerende sonder, som normal god landbrugspraksis-LC3, er i øjeblikket den mest udbredte tilgange25, den forbigående karakter af LC3-baseret signal og dets høje sats af falsk-positive signaler begrænse sin følsomhed for påvisning af mitophagy i cellerne26. En kombination af western blotting for at måle mitokondrie protein niveau, kvantificering af mitokondrie-DNA, og Fluorescens mikroskopi analyse til colocalize mitokondrier med enten autophagosomes eller lysosomer ville være en god tilgang til at vurdere mitophagy. Dog kræver kvantificering begrænsninger og mangel på bekvemmelighed af eksisterende metoder nye tilgange. Indførelsen af en ny pH-afhængige fluorescerende proteiner, mitokondrie målet Keima (mt-Keima), i høj grad forbedret evne til at detektere mitophagy27. Af sammensmeltning af mitokondrie-targeting sekvens af cytokrom c oxidase subunit VIII (COX VIII) i Keima, er mt-Keima rettet mod mitokondriets matrix. De store skift i excitation peak Keima fra 440 nm til 586 nm (svarende til pH 7 og pH 4, henholdsvis) kan udnyttes til at vurdere mitophagy med gode nænsomt i både in vitro- og i vivo eksperimenter28,29 . Endnu vigtigere, forårsager modstanden i mt-Keima til lysosomale forringelse integration af signalet fra mt-Keima i lysosomer, giver mulighed for nem kvantitativ måling af mitophagy aktivitet. Fluorescens ændringen, der opstår i mt-Keima kan analyseres ved hjælp af enten Konfokal mikroskopi eller flow flowcytometri28,29. En flow flowcytometri-baserede metode til at måle mitophagy aktiviteten ved hjælp af mt-Keima har imidlertid ikke ydet i detaljer til dato.
Her beskriver vi en detaljeret protokol for en flow flowcytometri-baserede teknik til at måle mitophagy aktiviteten i celler ved hjælp af mt-Keima. Selv om vi har her vist, at analysen af handelsstrømmen flowcytometri held opdaget CCCP-induceret mitophagy i HeLa celler udtrykte parkerin, kan denne teknik anvendes til en lang række celletyper.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REAGENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">poly-L-lysine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS202-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:133px;">Hyclone</td>
			<td style="width:119px;">SH30013.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293T</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CRL-3216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPTI-MEM&#160;</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbofect</td>
			<td style="width:133px;">Thermos scientific</td>
			<td style="width:119px;">R0531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.45 &#956;m syringe filter</td>
			<td style="width:133px;">sartorius</td>
			<td align="right" style="width:119px;">16555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CCL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polybrene</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puromycin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carbonyl cyanide m-chlorophenyl hydrazine (CCCP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2759</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypsin-EDTA</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD LSRFortessa</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSDIVA</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">FACSDIVA (v8.0.1)</td>
			<td></td>
		</tr>
	</tbody>
</table>

sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Mitophagy er en proces i selektiv fjernelse af beskadigede eller unødvendige mitokondrier ved hjælp af autophagy maskiner. Tætte forbindelser har fundet mellem defekte mitophagy og forskellige sygdomme hos mennesker, herunder neurodegenerative sygdomme, kræft og metaboliske sygdomme. Derudover har nylige undersøgelser vist, at mitophagy er involveret i normal cellulære processer, såsom differentiering og udvikling. Men den præcise rolle og molekylære mekanismer bag mitophagy kræver yderligere undersøgelse. Derfor er det kritisk at udvikle en robust og praktisk metode til at måle ændringer i mitophagy aktivitet. Her beskriver vi en detaljeret protokol til kvantitativt mitophagy virksomhed gennem flowcytometri ved hjælp af mitokondrier-målrettet fluorescerende protein Keima (mt-Keima). Dette flow flowcytometri assay kan analysere mitophagy aktivitet mere hurtigt og fornuftigt end konventionelle mikroskopi - eller immunoblotting-baserede metoder. Denne protokol kan anvendes til at analysere mitophagy aktivitet i forskellige celletyper.

Et eksempel på flow flowcytometri analyse af CCCP-induceret mitophagy i HeLa-Parkin celler er vist i figur 4. Ved hjælp af den flow flowcytometri analysemetode, der er beskrevet ovenfor, kan vi opdage en dramatisk stigning i mitophagic celler i den "høje" gate. Procentdelen af celler i den "høje" gate var steget mere end 10-fold sammenlignet med ubehandlet kontrol celler (figur 4A). Denne stigning i mitophagy aktivitet blev h...

Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, selektiv nedbrydningen af mitokondrier, har været impliceret i mitokondrie homøostase og er liberaliseret i forskellige sygdomme hos mennesker. Praktisk eksperimentelle metoder til at måle mitophagy aktiviteten er imidlertid meget begrænset. Her, leverer vi en følsom analyse til måling af mitophagy aktivitet ved hjælp af flowcytometri.

Følsomme måling af Mitophagy ved Flow flowcytometri ved hjælp af pH-afhængige fluorescerende Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Research

JoVE Journal

Biology

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Optimeret farvning og spredning modelleringsmetoder for celledeling Overvågning ved hjælp cellesporing Farvestoffer

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Immunodetektion af ydre membranproteiner ved flowcytometri Isoleret Mitokondrier

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Teknikker til analyse af ekstracellulær Vesikler anvendelse af flowcytometri

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Samtidig måling af Mitokondriel Calcium og mitokondriemembranen Potentiale i levende celler ved Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Måling af endoreduplikation ved flowcytometri af isolerede knold Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Høj overførselshastighed måling af Dictyostelium discoideum Macropinocytosis af Flow flowcytometri

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...