Name: monitoring stub1-mediated pexophagy
Uploaded: 2023-05-12T12:35:00
Description: Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

Dit werk werd gedeeltelijk ondersteund door een MOST 111-2311-B-001-019-MY3 onderzoeksbeurs van de National Science and Technology Council in Taiwan.

1. Bereiding van cellen die diKillerRed of zelflabelende eiwitten (SLPs) tot expressie brengen in het peroxisoomlumen
<ol><li>Zaai de gewenste cellen op celkweekschalen met glazen bodem. Voor het experiment hier, zaad 2 x 105 menselijke SHSY5Y-cellen in 840 μL kweekmedium (DMEM / F-12 aangevuld met 10% foetaal runderserum en 1% penicilline / streptomycine) of 6 x 104 muis NIH3T3-cellen in 840 μL kweekmedium (DMEM aangevuld met 10% runderserum en 1% penicilline / streptomycine) o...

<ol>
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M., Lodhi, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+dysfunction+in+age-related+diseases.">Peroxisomal dysfunction in age-related diseases.</a> Trends in Endocrinology &amp; Metabolism. 28 (4), 297-308 (2017).</li><li>Fransen, M., Nordgren, M., Wang, B., Apanasets, O., Veldhoven, P. P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+age-related+diseases+and+peroxisomes.">Aging, age-related diseases and peroxisomes.</a> Peroxisomes and their Key Role in Cellular Signaling and Metabolism. 69, 45-65 (2013).</li><li>Puri, P., 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=The+plasma+lipidomic+signature+of+nonalcoholic+steatohepatitis.">The plasma lipidomic signature of nonalcoholic steatohepatitis.</a> Hepatology. 50 (6), 1827-1838 (2009).</li><li>Law, K. B., 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=The+peroxisomal+AAA+ATPase+complex+prevents+pexophagy+and+development+of+peroxisome+biogenesis+disorders.">The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders.</a> Autophagy. 13 (5), 868-884 (2017).</li><li>Nazarko, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy+is+responsible+for+65%+of+cases+of+peroxisome+biogenesis+disorders.">Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders.</a> Autophagy. 13 (5), 991-994 (2017).</li><li>Eberhart, T., Kovacs, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy+in+yeast+and+mammals:+An+update+on+mysteries.">Pexophagy in yeast and mammals: An update on mysteries.</a> Histochemistry and Cell Biology. 150 (5), 473-488 (2018).</li><li>Manjithaya, R., Nazarko, T. Y., Farr&#233;, J. -. C., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+role+of+pexophagy.">Molecular mechanism and physiological role of pexophagy.</a> FEBS Letters. 584 (7), 1367-1373 (2010).</li><li>Till, A., Lakhani, R., Burnett, S. F., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy:+The+selective+degradation+of+peroxisomes.">Pexophagy: The selective degradation of peroxisomes.</a> International Journal of Cell Biology. 2012, 512721 (2012).</li><li>Germain, K., Kim, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy:+A+model+for+selective+autophagy.">Pexophagy: A model for selective autophagy.</a> International Journal of Molecular Sciences. 21 (2), 578 (2020).</li><li>Kim, P. K., Hailey, D. W., Mullen, R. T., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+signals+autophagic+degradation+of+cytosolic+proteins+and+peroxisomes.">Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (52), 20567-20574 (2008).</li><li>Yamashita, S., Abe, K., Tatemichi, Y., Fujiki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+peroxin+PEX3+induces+peroxisome-ubiquitination-linked+pexophagy.">The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy.</a> Autophagy. 10 (9), 1549-1564 (2014).</li><li>Nordgren, M., 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=Export-deficient+monoubiquitinated+PEX5+triggers+peroxisome+removal+in+SV40+large+T+antigen-transformed+mouse+embryonic+fibroblasts.">Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.</a> Autophagy. 11 (8), 1326-1340 (2015).</li><li>Zhang, 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=ATM+functions+at+the+peroxisome+to+induce+pexophagy+in+response+to+ROS.">ATM functions at the peroxisome to induce pexophagy in response to ROS.</a> Nature Cell Biology. 17 (10), 1259-1269 (2015).</li><li>Chen, B. -. H., Chang, Y. -. J., Lin, S., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsc70/Stub1+promotes+the+removal+of+individual+oxidatively+stressed+peroxisomes.">Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes.</a> Nature Communications. 11 (1), 5267 (2020).</li><li>Hung, Y. H., Chen, L. M., Yang, J. Y., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+controlled+induction+of+autophagy-mediated+lysosome+turnover.">Spatiotemporally controlled induction of autophagy-mediated lysosome turnover.</a> Nature Communications. 4, 2111 (2013).</li><li>Mageswaran, S. K., Yang, W. Y., Chakrabarty, Y., Oikonomou, C. M., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-electron+tomography+workflow+reveals+protrusion-mediated+shedding+on+injured+plasma+membrane.">A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.</a> Science Advances. 7 (13), eabc6345 (2021).</li><li>Yang, J. Y., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+controlled+initiation+of+parkin-mediated+mitophagy+within+single+cells.">Spatiotemporally controlled initiation of parkin-mediated mitophagy within single cells.</a> Autophagy. 7 (10), 1230-1238 (2011).</li><li>Yang, J. Y., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bit-by-bit+autophagic+removal+of+parkin-labelled+mitochondria.">Bit-by-bit autophagic removal of parkin-labelled mitochondria.</a> Nature Communications. 4, 2428 (2013).</li><li>Los, G. V., 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=HaloTag:+A+novel+protein+labeling+technology+for+cell+imaging+and+protein+analysis.">HaloTag: A novel protein labeling technology for cell imaging and protein analysis.</a> ACS Chemical Biology. 3 (6), 373-382 (2008).</li><li>Lismont, C., Walton, P. A., Fransen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+subcellular+redox+dynamics+in+living+mammalian+cells+using+RoGFP2-based+probes.">Quantitative monitoring of subcellular redox dynamics in living mammalian cells using RoGFP2-based probes.</a> Methods in Molecular Biology. 1595, 151-164 (2017).</li><li>Nordgren, M., 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=Potential+limitations+in+the+use+of+KillerRed+for+fluorescence+microscopy.">Potential limitations in the use of KillerRed for fluorescence microscopy.</a> Journal of Microscopy. 245 (3), 229-235 (2012).</li></ol>

Dit protocol beschrijft hoe Stub1-gemedieerde pexofagie in celculturen kan worden geactiveerd door peroxisomale ROS-niveaus met licht te verhogen. Omdat het protocol afhankelijk is van kleurstof-geassisteerde ROS-generatie, moet men zorgen voor voldoende expressie van diKillerRed-VKSKL of kleurstof-gelabelde SLP-ligandkleuring in de cellen van belang. Gezien het feit dat verschillende celtypen of cellen met verschillende genetische achtergronden peroxisomen met enigszins verschillende eigenschappen kunnen bevatten, moet ...

Peroxisomen zijn enkelmembraangebonden organellen die aanwezig zijn in de meeste eukaryote cellen. Peroxisomen zijn een metabolisch compartiment dat essentieel is voor het uitvoeren van de bèta-oxidatie van vetzuren met een zeer lange keten, purinekatabolisme en etherfosfolipide- en galzuursynthese1. Peroxisoom-afgeleid acetyl-CoA regelt lipidehomeostase door de centrale signalering in metabolisme2 te reguleren. Daarom is het geen verrassing dat gecompromitteerde peroxisomale functies worden geïmpliceerd in verschillende ziekten, waaronder neurodegeneratieve aandoeningen, veroudering, kanker, obesitas en diabetes 3,4,5. Een essentieel proces bij het handhaven van peroxisomale werking is pexofagie. Pexofagie is een katabolisch proces voor de selectieve omzetting van peroxisomen door autofagie. Cellen gebruiken pexofagie om de kwantiteit en kwaliteit van peroxisomen te helpen beheersen, waardoor een goede peroxisomale functie wordt gegarandeerd. Een recente studie toonde aan dat peroxisoomverlies veroorzaakt door mutaties in PEX1 peroxisomale biogenese factoren 1 en 6 het gevolg is van ongecontroleerde pexofagie6. Met name 65% van alle patiënten met peroxisoombiogenesestoornis (PBD) heeft tekortkomingen in het peroxisomale AAA ATPase-complex, bestaande uit PEX1, PEX6 en PEX26 in zoogdiercellen7.
Een aantal methoden kan worden gebruikt om pexofagie te initiëren en te bestuderen. In gist wordt pexofagie geactiveerd wanneer de geleverde voedingsstoffen worden overgeschakeld van peroxisoomafhankelijke koolstofbronnen naar peroxisoomonafhankelijke koolstofbronnen (naar lagere cellulaire peroxisoomaantallen)8. De overdracht van methanol-gekweekte Pichia pastoris-cellen van methanolmedium naar glucosemedium en ethanolmedium induceert bijvoorbeeld micropexofagie en macropexofagie, respectievelijk 8,9,10. Micropexofagie-sequesters clusterden peroxisomen voor afbraak door de vacuole te hermodelleren om cup-achtige vacuolaire sekwestratiemembranen en een dekselachtige structuur te vormen die het micropexofagie-specifieke membraanapparaat (MIPA) wordt genoemd. Bij macropexofagie worden individuele peroxisomen overspoeld door dubbelmembraanstructuren die bekend staan als pexofagosomen, gevolgd door fusie met de vacuole voor afbraak 8,9,10. De fosforylering van pexofagiereceptoren, zoals Atg36p in Saccharomyces cerevisiae en Atg30p in Pichia pastoris, is van cruciaal belang voor de receptoren om kernautofagiemachines te rekruteren en om peroxisomale targeting op autofagosomen 8,11 te vergemakkelijken.
In zoogdiercellen kan pexofagie worden geïnduceerd door ubiquitinatie. Het labelen van de peroxisomale membraaneiwitten PMP34 of PEX3 met ubiquitine aan de cytosolische kant induceert pexofagie12. De overexpressie van PEX3 induceert peroxisoom ubiquitinatie en peroxisoom eliminatie door lysosomen13. Bovendien schaadt de fusie van PEX5 met een C-terminale EGFP de export van monoubiquitinated PEX5 en resulteert in pexofagie14. Aan de andere kant kan pexofagie ook worden veroorzaakt door H 2 O2-behandeling. Peroxisomen produceren reactieve zuurstofsoorten (ROS); specifiek produceert het peroxisomale enzym Acox1, dat de eerste stap van bèta-oxidatie van zeer lange keten vetzuren (> 22 koolstof) katalyseert, niet alleen acetyl-CoA, maar ook peroxisomale ROS. Als reactie op de verhoogde ROS-niveaus onder H 2 O2-behandelingactiveren zoogdiercellen pexofagie om de ROS-productie te verlagen en stress te verlichten. Er is gemeld dat H 2 O2-behandelingde rekrutering van ataxie-teleangiëctasieën gemuteerd (ATM) naar peroxisomen stimuleert. ATM fosforyleert vervolgens PEX5 om de peroxisomale omzet te bevorderen door pexofagie15.
Omdat peroxisomen ROS-genererende centra zijn, zijn ze ook gevoelig voor ROS-schade. ROS-uitgelokte peroxisomale letsels dwingen cellen om pexofagie te activeren om peroxisoomkwaliteitscontroleroutes te initiëren (verwijdering van het beschadigde peroxisoom door autofagie). Hier schetsen we een aanpak voor het on-demand triggeren van ROS-uitgelokt peroxisomale schade. Het protocol maakt gebruik van lichtgeactiveerde ROS-productie binnen organellen 16,17,18,19,20 (figuur 1). Peroxisomen met kleurstoflabel worden verlicht, wat leidt tot ROS-productie in het peroxisomale lumen, wat specifiek peroxisomale schade veroorzaakt. Met behulp van dit protocol wordt aangetoond dat ROS-gestresste peroxisomen worden verwijderd via een ubiquitine-afhankelijke afbraakroute. ROS-gestreste peroxisomen rekruteren het ubiquitine E3-ligase Stub1 om hun overspoeling in autofagosomen mogelijk te maken voor individuele verwijdering door pexofagie16. Men kan dit protocol gebruiken om het lot van gewonde en gezonde peroxisomen in dezelfde cel te vergelijken door middel van time-lapse microscopie. De methode kan ook worden gebruikt om alle peroxisomen (in alle cellen) op een kweekschaal globaal te beschadigen, waardoor de biochemische analyse van de pexofagieroute mogelijk is.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm culture dish with a 20 mm diameter glass microwell&#160;</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>20 mm glass bottomed</td>
		</tr>
		<tr>
			<td>3-amino-1,2,4-triazole (3-AT)</td>
			<td>Sigma Aldrich</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>16170060</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Nuaire</td>
			<td>NU-4750</td>
			<td></td>
		</tr>
		<tr>
			<td>diKillerRed-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>made by appending the KillerRed tandem dimer with PTS1(VKSKL)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)</td>
			<td>ThermoFisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-C1</td>
			<td>Clontech</td>
			<td>pEGFP-C1</td>
			<td>The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62</td>
		</tr>
		<tr>
			<td>EGFP-Hsp70</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>Hsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-LC3B</td>
			<td>Addgene</td>
			<td>11546</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-p62</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Stub1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Ub</td>
			<td>Addgene</td>
			<td>11928</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag TMR ligand&#160;</td>
			<td>Promega</td>
			<td>G8252</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PTS1 appended and cloned into EGFP-C1 backbone</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Confocal Microscope&#160;</td>
			<td>Olympus</td>
			<td>FV3000RS</td>
			<td>405 nm Ex, 488 nm Ex, 561 nm Ex,&#160; microscope with a TOKAI HIT chamber incubator and the UNIV2-D35 dish attachment</td>
		</tr>
		<tr>
			<td>Janelia Fluor 646 HaloTag Ligand</td>
			<td>Promega</td>
			<td>GA1120</td>
			<td></td>
		</tr>
		<tr>
			<td>LED</td>
			<td>VitaStar</td>
			<td>PAR64</td>
			<td>80 W, 555-570 nm</td>
		</tr>
		<tr>
			<td>lipofectamine 2000&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11668</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>NIH3T3 cell</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td>adherent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>319850</td>
			<td>reduced serum media</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMP34-TagBFP</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171)</td>
		</tr>
		<tr>
			<td>roGFP2-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1</td>
		</tr>
		<tr>
			<td>SHSY5Y cell</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
			<td>adherent</td>
		</tr>
	</tbody>
</table>

monitoring stub1-mediated pexophagy

Zoogdiercellen kunnen peroxisomen omkeren via Stub1-gemedieerde pexofagie. De route maakt mogelijk cellulaire controle van de kwantiteit en kwaliteit van peroxisomen mogelijk. Tijdens dit proces transloceren heat shock protein 70 en het ubiquitine E3 ligase, Stub1, op peroxisomen om te worden omgedraaid om pexofagie te initiëren. De Stub1 ligase-activiteit maakt de accumulatie van ubiquitine en andere autofagie-gerelateerde modules op gerichte peroxisomen mogelijk. Het verhogen van reactieve zuurstofsoorten (ROS) niveaus binnen het peroxisomale lumen kan Stub1-gemedieerde pexofagie activeren. Men kan daarom kleurstof-ondersteunde ROS-generatie gebruiken om deze route te activeren en te monitoren. Dit artikel schetst de procedures voor het gebruik van twee klassen kleurstoffen, fluorescerende eiwitten en synthetische fluoroforen, om pexofagie in zoogdiercelculturen te initiëren. Deze op kleurstof ondersteunde ROS-generatie gebaseerde protocollen kunnen niet alleen worden gebruikt om alle peroxisomen binnen een celpopulatie wereldwijd aan te pakken, maar kunnen ook de manipulatie van individuele peroxisomen binnen afzonderlijke cellen mogelijk maken. We beschrijven ook hoe Stub1-gemedieerde pexofagie kan worden gevolgd met behulp van live-cell microscopie.

Het hier getoonde Stub1-gemedieerde pexofagie-inductieschema maakt gebruik van kleurstofondersteunde ROS-generatie binnen het peroxisoomlumen. Deze bewerking vereist minimale lichtintensiteiten. Peroxisomen die fluorescerende eiwitten of kleurstoffen bevatten, kunnen daarom worden verlicht met behulp van standaard laserscannende confocale microscopen. Focale verlichting leidt tot onmiddellijke en gelokaliseerde ROS-productie binnen individuele peroxisomen, zoals aangegeven door de fluorescerende reporter roGFP2-VKSKL (<s...

Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

Monitoring Stub1-Mediated Pexophagy

Dit protocol geeft instructies voor het activeren en monitoren van Stub1-gemedieerde pexofagie in levende cellen.

Monitoring Stub1-gemedieerde Pexofagie

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality ...

Peroxisomen voeren bèta-oxidatie uit en zijn gevoelig voor ROS-schade. Deze methode maakt het mogelijk om te onderzoeken hoe cellen omgaan met ROS-gestresste peroxisomen. Dit protocol wordt niet alleen gebruikt om zich wereldwijd te richten op alle peroxisomen binnen een celpopulatie, maar kan ook de manipulatie van de individuele peroxisomen binnen afzonderlijke cellen mogelijk maken.Kleurstof-geassisteerde ROS-generatie kan ook worden gebruikt om organellen buiten peroxisomen te richten om te onderzoeken hoe cellen omgaan met organelletsels. Om te beginnen, zaai twee keer 10 tot de vijfde menselijke SHSY5Y-cellen of zes keer 10 tot de vierde muis NIH H3T3-cellen in 840 microliter kweekmedium op 35-millimeter kweekschalen met glazen bodem met een glazen microwell met een diameter van 20 millimeter. Kweek de cellen onder 5% koolstofdioxide bij 37 graden Celsius gedurende 24 uur.Transfecteer vervolgens de cellen met de gewenste plasmiden met behulp van een transfectiereagens of SHSY5Y-celtransfectie. Verdun plasmiden in 55 microliter serumvrij DMEM/F-12 en verdun één microliter transfectiereagens in 55 microliter serumvrij DMEM/F-12. Combineer het verdunde DNA met het verdunde reagens.Meng voorzichtig en incubeer gedurende 30 minuten bij kamertemperatuur. Voeg het complex toe aan de 20 millimeter microwell die eerder is bedekt met 100 microliter kweekmedium dat SHSY5Y-cellen zonder antibiotica bevat. Schud de schaal voorzichtig heen en weer.Voer NIH 3T3-celtransfectie uit zoals eerder aangetoond, maar vervang gereduceerd serummedium in plaats van serumvrije DMEM / F-12 voor plasmideverdunning en transfectiereagens. Verwijder na twee uur transfectie het transfectiereagensbevattende medium en voeg één milliliter vers kweekmedium toe. Incubeer de NIH 3T3- of SHSY5Y-cellen bij 37 graden Celsius en 5% koolstofdioxide gedurende 24 uur.Voor lichtgeactiveerde ROS-productie verwijdert u 24 uur na transfectie het kweekmedium en voegt u 10 microliter 200 nanomolair HaloTag TMR-ligand of 200 nanomolair Janelia Fluor 646 HaloTag-ligand toe aan de 20-millimeter glazen microwell. Breng de schaal over naar een 37 graden Celsius en 5% koolstofdioxide-incubator en laat het een uur staan om de cellen te kleuren. Verwijder na een uur het kleurmedium grondig en voeg een milliliter vers kweekmedium toe.Plaats een schaal met glazen bodem met de gewenste cellen op een laserscanning confocale microscoop uitgerust met een podiumincubator. Klik in de software op Gereedschapsvenster, kies Oculair en klik op de DIA-knop om het doorgelaten licht in te schakelen. Bekijk de schotel door het microscoopoculair en breng de cellen scherp door aan de scherpstelknop te draaien.Kies LSM en klik op de knop Dye Detector Select. Dubbelklik op de gewenste kleurstoffen in het pop-upvenster om de juiste laser- en filterinstellingen te selecteren voor het afbeelden van de cellen. Klik op Live4x in het Live-paneel om een snelle laserscanning te starten om te beginnen met het screenen op de fluorescerende signalen van de cellen.Verplaats het podium met behulp van de joystickcontroller en zoek de medium diKillerRed VKSKL- of SLP-VKSKL-expresserende cellen om ROS-spanning op de peroxisomen toe te passen. Verkrijg een afbeelding van de gewenste cel. Klik op Tool Window en kies LSM Stimulation.Klik vervolgens op de ellipsknop en gebruik in het Live Image-venster de muis om een ROI van 15 micrometer in diameter te tekenen met de gewenste peroxisomen waarop ROS-spanning zal worden toegepast. Om ROS-stress toe te passen, gebruikt u 561 nanometer voor cellen met de diKillerRed VKSKL of TMR-gelabelde SLP-ligand en gebruikt u 640 nanometer voor cellen gekleurd met de Janelia Fluor 646-gelabelde SLP-ligand. Selecteer de lasergolflengte voor het toepassen van de ROS-spanning.Voer het gewenste laserpercentage en de duur in. Voor ROS-productie in de peroxisomen klikt u op Acquire en vervolgens op de stimulatieknop om het scannen te starten met 561 of 640 nanometer licht door de ROIs gedurende elk 30 seconden. Deze stimulatie komt overeen met ongeveer 5% laservermogensinstellingen voor 561 en 640 nanometer.Na 30 seconden laserverlichting verliezen peroxisomen binnen de ROIs hun diKillerRed VKSKL-, TMR- of Janelia Fluor 646-signalen. Dit signaalverlies maakt het mogelijk om de verlichte en niet-verlichte peroxisomen in de cel te onderscheiden. Om de pexofagean levende cellen te controleren, volgt u de translocatie van EGFP-gelabelde Stub1, Hsp70, ubiquitine, p62 of LC3B op de verlichte of ROS-belaste peroxisomen door timelapse-beeldvorming met intervallen van 10 minuten tussen frames.Focale verlichting leidt tot onmiddellijke en gelokaliseerde ROS-productie binnen individuele peroxisomen zoals aangegeven door de fluorescerende reporter roGFP2-VKSKL. De diKillerRed VKSKL-afbeelding werd genomen nadat alle roGFP2-VKSKL-metingen waren gedaan om de locatie van de beschadigde peroxisomen aan te geven. De gegevens tonen ook aan dat niet-geanolumineerde peroxisomen niet worden beïnvloed, wat wijst op de precisie van de methodologie.Deze methodologie werd gebruikt om Stub1-gemedieerde pexofagie te monitoren. Tijdens dit proces verschijnen Hsp70, Stub1, ubiquitinated proteins, autofagy adapters p62 en LC3B vervolgens in ROS-gestresste peroxisomen om pexofagie aan te drijven. Met dezelfde strategie was het mogelijk om tegelijkertijd alle peroxisomen op een kweekschaal te beschadigen voor de biochemische karakterisering van Stub1-gemedieerde pexofagie.Vóór LED-verlichting was EGFP-ubiquitinefluorescentie homogeen in het cytoplasma en colokaliseerde het niet met de peroxisomen. Na negen uur LED-verlichting hoopten de EGFP-ubiquitinesignalen zich op op alle peroxisomen in de cellen die in een confocale schaal werden gekweekt. Met behulp van dit protocol ontdekten we dat ROS-gestresste peroxisomen worden verwijderd via een ubiquitine-afhankelijke afbraakroute.ROS-gestreste peroxisomen rekruteren het ubiquitine E3-ligase Stub1 om hun verwijdering door pexofagie mogelijk te maken.

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoring Actin Demontage met Time-lapse Microscopie

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Functionele Beeldvorming met Wapening, Eyetracking, en Fysiologische Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Single Molecule methoden voor monitoring van veranderingen in bilaag elastische eigenschappen

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Monitoring plantenhormonen tijdens stress Reacties

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Monitoring Acupunctuur effecten op de menselijke hersenen door fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Monitoring Heart Functie in larvale Drosophila melanogaster Voor Fysiologische Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Bioassays voor Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Lange-termijn Lethal toxiciteit Test met de Schaal- Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Gekoppeld Testen voor Monitoring Protein Refolding in<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Geselecteerde Reaction Monitoring Massaspectrometrie voor Absolute Protein kwantificering

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...