Name: quantitative analysis of autophagy using advanced 3d fluorescence microscopy
Uploaded: 2013-05-03T12:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy at JoVE.com

Grant support: NIH CA165263, NIH CA150197, NIH CA150197S1 (HJ Kung), NIH CA150197S1 (CA Changou), NSF PHY-0.120.999 Center for Biophotonics Science &amp; Technology (DL Wolfson, FYS Chuang), DOD PC073420 (RJ Fed), The Research Norges, Leiv Eiriksson rejselegat 209286/F11 (BS Ahluwalia). HJ Kung anerkender også støtte fra Auburn Community Cancer Endowment Fund. RJ Fed anerkender også støtte fra J. McDonald begavelse. Vi takker Dr. Jenny Wei-Jen Kung og Dr. Bor-wen Wu på DesigneRx for generøs levering af ADI.

1.. Del 1: Cellekultur og immuno-fluorescerende mærkning <ol><li> Grow CWR22 RV1 humane prostata tumorceller på dækglas (# 1.5 eller 170 um tykkelse) anbragt i 6-brønds plader, med RPMI (Mediatech, VA) indeholdende 10% kalvefosterserum (FBS) og 1% penicillin / streptomycin / glutamin. </li><li> Fremkalde autophagy ved behandling udvalgte prøver med arginindeiminase (ADI, 0,3 ug / ml) i phosphatpufret saltvand (PBS). </li><li> Lave celler med 4% paraformaldehyd (PFA, Fisher Scientific, NH) fortyndet i PBS...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>Kabeya, Y., Mizushima, N., Ueno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+a+mammalian+homologue+of+yeast+Apg8p,+is+localized+in+autophagosome+membranes+after+processing.">LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.</a> EMBO J. 19, 5720-5728 (2000).</li><li>Pattingre, S., Espert, L., Biard-Piechaczyk, M., Codogno, P. <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+macroautophagy+by+mTOR+and+Beclin+1+complexes.">Regulation of macroautophagy by mTOR and Beclin 1 complexes.</a> Biochimie. 90, 313-323 (2008).</li><li>Maiuri, M. C., Criollo, A., Tasdemir, 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=BH3-only+proteins+and+BH3mimetics+induce+autophagy+by+competitively+disrupting+the+interaction+between+Beclin+1+and+Bcl-2/Bcl-X(L).">BH3-only proteins and BH3mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).</a> Autophagy. 3, 374-376 (2007).</li><li>Crighton, D., Wilkinson, S., O'Prey, 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=DRAM,+a+p53-induced+modulator+of+autophagy,+is+critical+for+apoptosis.">DRAM, a p53-induced modulator of autophagy, is critical for apoptosis.</a> Cell. 126, 121-134 (2008).</li><li>Dillon, B. J., Prieto, V. G., Curley, S. 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Mens de direkte observation af celler mærket med fluorescerende prober mod LC3 almindeligt accepteret som en standard metode til at bekræfte autophagic respons 6, kvantitativ 3D billeddannelse af det samme system (som vi har gjort) giver hidtil uset information og detaljer om den komplekse proces cellulær autophagy . I særdeleshed, observere vi, at hundredvis (hvis ikke tusindvis) af autophagosomes i levende celler er dannet inden for 80 min af autophagy induktion. Ligeledes ser vi meget interessante morf...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Arginine Deiminase (ADI)</td>
			<td>DesigneRx</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C5890</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>4042</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S4521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-mouse 555</td>
			<td>Invitrogen</td>
			<td>A21422</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-rabbit 647</td>
			<td>Invitrogen</td>
			<td>A21244</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LysoTracker Red DND-99</td>
			<td>Invitrogen</td>
			<td>L7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Lamp1</td>
			<td>DSHB</td>
			<td>H4A3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Cadherin</td>
			<td>Cell Signaling</td>
			<td>#3195</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlowFade Gold</td>
			<td>Invitrogen</td>
			<td>S36936</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-d-lysine coated glass bottom plate</td>
			<td>MatTek</td>
			<td>P35GC-1.5-1.4-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>No.1, 22 mm coverslip</td>
			<td>Corning</td>
			<td>#2865-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Globe Scientific</td>
			<td>1324G</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

quantitative analysis of autophagy using advanced 3d fluorescence microscopy

Prostatakræft er den førende form af maligne sygdomme blandt mænd i USA Mens kirurgi indebærer en væsentlig risiko for impotens og inkontinens, har traditionelle kemoterapeutiske tilgange stort set været forgæves. Hormonbehandling er effektiv på et tidligt tidspunkt, men ofte mislykkes med den endelige udvikling af hormon-ildfaste tumorer. Vi har været interesseret i at udvikle lægemidler rettet mod specifikke metaboliske mangel af tumorceller. Vi har for nylig vist, at prostata tumorceller specifikt mangler et enzym (argininosuccinate syntase, eller ASS) involveret i syntesen af aminosyren arginin 1.. Denne betingelse forårsager tumorcellerne at blive afhængig af eksogene arginin, og de ​​gennemgår metabolisk stress, når fri arginin er forårsaget af arginindeiminase (ADI) 1,10. Vi har faktisk vist, at humane prostata cancer celler CWR22 RV1 effektivt dræbt af ADI med caspase-uafhængig apoptose og aggressive autophagy (eller macroautophagy) 1,2,3. Autophagy er en evolutionært-bevarede proces, der tillader celler at metabolisere uønskede proteiner ved lysosomal nedbrydning under ernæringsmæssige sult 4,5. Selvom de væsentlige elementer i denne vej er godt præget 6,7,8,9, mange aspekter af den molekylære mekanisme er stadig uklart - i særdeleshed, er hvilken rolle autophagy i død-respons af prostatakræft celler efter ADI behandling ? For at løse dette spørgsmål, vi krævede en eksperimentel metode til at måle niveauet og omfanget af autophagic respons i celler - og da der er ingen kendte molekylære markører, der kan præcist spore denne proces, valgte vi at udvikle en imaging tilgang, ved hjælp af kvantitativ 3D fluorescensmikroskopi 11,12. Brug CWR22Rv1 celler specifikt-mærket med fluorescerende prober til autophagosomes og lysosomer, viser vi, at 3D billedstakke erhverves med enten widefield deconvolution mikroskopi (og senere, med super-opløsning, struktureret-belysning mikroskopi) kan tydeligt fange de tidlige stadier af autophagy induktion. Med kommercielt tilgængelige digitale billedanalyse applikationer, kan vi let få statistiske oplysninger om autophagosome og lysosom antal, størrelse, distribution, og graden af ​​colokalisering fra enhver afbildet celle. Denne information giver os mulighed for præcist at spore fremskridt af autophagy i levende celler og giver vores fortsatte undersøgelse af rolle autophagy i cancer kemoterapi.

Billedet sekvensen vist i Figur 1 viser de fysiske forandringer, der sker i CWR22-celler i løbet af de første 80 min af autophagy induktion. I denne og andre undersøgelser (ikke vist) vi konsekvent overholdes: (1) forskydning af kernen bort fra midten af ​​cellen, (2) reduktion af Focal berøringspunkter, og (3) generelle translokation af autophagosomes og lysosomer mod midten af celle. Desuden observerede vi en lille stigning i colokalisering (med gult) mellem autophagosomes (grøn) og lysosomer...

Watch this Scientific Journal Video about Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy at JoVE.com

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Autophagy er en allestedsnærværende proces, der gør det muligt for cellerne at nedbryde og genbruge proteiner og organeller. Vi anvender avanceret fluorescens mikroskopi til at visualisere og kvantificere små, men vigtige, fysiske ændringer i forbindelse med induktion af autophagy, herunder dannelse og distribution af autophagosomes og lysosomer, samt deres fusion i autolysosomes.

Kvantitativ analyse af Autophagy hjælp Advanced 3D fluorescensmikroskopi

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

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