Name: assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans
Uploaded: 2018-04-13T14:30:00
Description: Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Vi vil gerne takke Caenorhabditis genetik Center for stammer, naturvidenskab og teknik forskning Rådet (NSERC) og Canada Foundation for Innovation (CFI) for finansiering. Vi vil gerne takke Dr. Lizhen Chen (Institut for celle systemer og anatomi, UT sundhed San Antonio) for at tillade ubegrænset brug af hendes laboratoriefaciliteter for alle C. elegans eksperimenter samt Dr. Exing Wang (Associate Director, optisk tænkelig facilitet UT sundhed San Antonio) for assistance med Konfokal mikroskopi. Vi vil også gerne takke Dr. Myron Ignatius for at yde støtte og opmuntring til at lette den video shoot.

1. pletten og billed intestinal lysosomer
<ol><li>Frø ødelægge vækst medier (NGM) plader med OP50
	<ol>
<li>Forberede NGM plader pr anbefalede protokol19 og tillade lukkede pladerne tørre i 2 dage ved stuetemperatur.</li>
		<li>Podes OP50 bakterier i sterile Luria bouillon (LB) bouillon og vokse i en rystende inkubator eller vand bad ved 37 ° C i 36 timer, eller indtil OD er mellem 0,2 og 0,4. Undgå at bruge en bakteriekultur med OD > 1 eller OD < 0,2.</li>
		<li>Når NGM...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=btn1,+the+Schizosaccharomyces+pombe+homologue+of+the+human+Batten+disease+gene+CLN3,+regulates+vacuole+homeostasis.">btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis.</a> J Cell Sci. 118 (Pt 23), 5525-5536 (2005).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Preston, R. A., Murphy, R. F., Jones, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+vacuolar+pH+in+yeast+and+identification+of+acidification-defective+mutants.">Assay of vacuolar pH in yeast and identification of acidification-defective mutants.</a> Proc Natl Acad Sci U S A. 86 (18), 7027-7031 (1989).</li><li>Pringle, J. R., 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=Fluorescence+microscopy+methods+for+yeast.">Fluorescence microscopy methods for yeast.</a> Methods Cell Biol. 31, 357-435 (1989).</li><li>Baxi, K., Ghavidel, A., Waddell, B., Harkness, T. A., de Carvalho, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Lysosomal+Function+by+the+DAF-16+Forkhead+Transcription+Factor+Couples+Reproduction+to+Aging+in+Caenorhabditis+elegans.">Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.</a> Genetics. 207 (1), 83-101 (2017).</li><li>Colacurcio, D. J., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+lysosomal+acidification-The+emerging+role+of+v-ATPase+in+aging+and+neurodegenerative+disease.">Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease.</a> Ageing Res Rev. 32, 75-88 (2016).</li><li>Kang, H. 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=Chemical+screening+identifies+ATM+as+a+target+for+alleviating+senescence.">Chemical screening identifies ATM as a target for alleviating senescence.</a> Nat Chem Biol. 13 (6), 616-623 (2017).</li><li>Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+formation+reduces+levels+of+mutant+huntingtin+and+the+risk+of+neuronal+death.">Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.</a> Nature. 431 (7010), 805-810 (2004).</li><li>Brunk, U. 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En bred vifte af cellulære og molekylære begivenheder bidrage til aldring, påvirket af livshistorie træk og genetiske faktorer. Vores seneste undersøgelse22 tyder på, at den reproduktive cyklus spiller en vigtig rolle i at kontrollere fitness af soma gennem regulering af lysosomale pH dynamics. Vi viste, at lysosomale-medieret proteolyse fremmes mens dyr aktivt reproducere ved opregulering af v-ATPase transskription, som igen sikrer sure lysosomer. Ved slutningen af reproduktion, v-ATPase ud...

Forekomsten af protein aggregater er almindeligt accepteret for at være kendetegnende for aldring i eukaryote celler1,2,3, og den dannelse, som menes at være blandt princippet førerne af cellulære gulne4 , 5 , 6 , 7. der er stigende tegn, som cellerne alder, protein katabolisme er svækket, fører til en stigning i protein sammenlægning. Sammenbruddet af proteolyse i aging celler indebærer en forringelse af autophagy8 samt proteasom-medieret protein nedbrydning9. Endelig, uigenkaldelig protein oxidation er steget i gamle celler, yderligere forværrer protein katabolisme10.
Autophagy var oprindeligt menes at være en ikke-selektiv proces for bulk nedbrydning af beskadigede proteiner, men nylige undersøgelser har vist, at autophagy er yderst selektiv til katabolisme af protein aggregater og dysfunktionelle organeller, der ikke er modtagelig for nedbrydning via andre protein clearance mekanismer11. Under processen med autophagy, er beskadiget og aggregerede proteiner afsondret i en dobbelt-membran vesikel kaldes autophagosome. Denne autophagosome derefter sikringer med de sure organeller kaldet lysosomer, hvilket fører til nedbrydning af autophagosome last12. Lysosomer repræsenterer farveprøve af den autophagic vej og deltage i forskellige cellulære processer såsom membran reparation, transcriptional kontrol og næringsstof sensing; fremhæve deres centrale rolle i cellulære homeostase (gennemgik i ref. 13). Flere undersøgelser har vist en tilknytning mellem en aldersbetinget fald i lysosomale funktion og forskellige neurodegenerative lidelser13. Konsekvent, kan genopretning lysosomale funktion i ældre celler sinke udviklingen af aldersrelaterede fænotyper14,15. Undersøgelser af sammensætningen af intralumen miljø tyder på, at sammenbruddet af lysosomale funktion i ældre celler ikke er på grund af en reduktion i produktionen af lysosomale proteaser16. Alternativt er det blevet foreslået, at tab af intralysosomal syreindhold, en kritisk kravet om dets enzymatiske aktivitet, kan ligge til grund for faldet i lysosomet-medieret proteolyse17. Hvis du vil udforske denne hypotese, er det vigtigt at udvikle reagenser og protokoller til sonde dynamiske ændringer i lysosomale pH i levende celler i en replikerbar og konsekvent måde.
Tarmen af C. elegans er den store metaboliske væv i orme og det er en kritisk regulator af systemisk homøostase og levetid. Vi har udviklet assays til at vurdere ændringer i surhedsgrad af lumen af intestinal lysosomer af orme til at bestemme, hvordan lysosomet-medieret proteolyse bidrager til ældning. Selv om pH-følsomme fluorophores har været brugt tidligere i C. elegans for at markere intestinal lysosomer, ikke har der endnu været et forsøg på at etablere en vellykket protokol, der kan detektere små stigninger i lysosomale pH i vivo18. Her, leverer vi en protokol, der kan bruges til at registrere tab af lysosomale surhedsgrad i tarmcellerne af C. elegans ved hjælp af en enkel og praktisk fodring protokol, der inkorporerer en pH-følsomme fluorophore (cDCFDA) i OP50 mad.

<table border="0" cellpadding="0" cellspacing="0" width="1166">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">OP50 (E. coli)</td>
			<td style="width:188px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td style="width:385px;">Order online at https://cgc.umn.edu/strain/OP50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">5(6)-carboxy-2&#8217;,7&#8217;-dichlorofluorescein diacetate&#160;</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">C369</td>
			<td>Commonly known as cDCFDA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:475px;">9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">L7528</td>
			<td>Commonly known as Lysotracker Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Confocal microscope (e.g. Zeiss LSM 510)</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">ImageJ</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td>Download for free from https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">LB Broth powder</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">22700041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Agar</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">NaCl</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Peptone</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:119px;">S71604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cholesterol powder</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">C3045&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">CaCl2</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">449709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">MgSO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">K3PO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">P5629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sodium Azide</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DMSO</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:119px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cover Slips</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">3406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Agarose</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Incubator</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mirror or other smooth flat surface</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

Nematode Caenorhabditis elegans (C. elegans) er en modelsystem, som er meget udbredt at studere levetiden og udviklingsmæssige veje. Sådanne undersøgelser er lettet af gennemsigtighed af dyret, evnen til at frem og bak genetiske assays, den relative lethed af generere fluorescently mærket proteiner og brug af fluorescerende farvestoffer, der kan enten være microinjected i den tidlige embryo eller indarbejdet i sin mad (E. coli stamme OP50) at mærke cellulære organeller (f.eks. 9-indføres-5 H-benzo (a) phenoxazine-5-one og (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-yl-kappaN} - N - [2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Vi præsenterer her, brugen af en fluorescerende pH-følsom farvestof, der pletter intestinal lysosomer, giver en visuel udlæsning af dynamiske, fysiologiske ændringer i lysosomale surhedsgrad i levende orme. Denne protokol måle ikke lysosomale pH, men snarere har til formål at etablere en pålidelig metode til vurdering af fysiologiske relevante varianter i lysosomale surhedsgrad. cDCFDA er en celle-permeant stof, der er konverteret til den fluorescerende fluorophore 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) ved hydrolyse af intracellulære esteraser. Protonation inde lysosomer fælder cDCF i disse organeller, hvor det ophobes. På grund af sin lave pKa af 4.8, har dette farvestof været brugt som en pH sensor i gær. Her beskriver vi brugen af cDCFDA som et kosttilskud at vurdere surhedsgraden i tarm lysosomer i C. elegans. Denne teknik giver mulighed for påvisning af alkalinizing lysosomer med levende dyr, og har en bred vifte af eksperimentelle applikationer, herunder undersøgelser af befolkningens aldring, autophagy og lysosomale Biogenese.

cDCFDA pletter lysosomer på en måde, pH-afhængige, og dens lave pKa og klar optagelse i lysosomer gør det en ideel pH sensor21. cDCFDA farvning intensitet er omvendt proportional med lysosomale pH (dvs . farvning af intensitet øges når pH falder)18,22. cDCFDA signaler er konsekvent svage lysosomer dyr behandlet med 20 mM af chloroquin, en hæmmer af lysosomale forsuring og orme forarmet af v-...

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Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

En trinvis vejledning til sonde tab af lysosomale surhedsgrad i tarmen af C. elegans brug af pH-følsom vitale farvestoffet 5 (6) - carboxy - 2', 7'-dichlorofluorescein diacetat (cDCFDA)

Vurdering af lysosomale basisk i tarmen af Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

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At studere Oxidative Stress forårsaget af Mitis gruppe streptokokker i Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

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Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...