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

Dette arbeidet ble delvis støttet av et MOST 111-2311-B-001-019-MY3 forskningsstipend fra National Science and Technology Council i Taiwan.

1. Fremstilling av celler som uttrykker diKillerRed eller selvmerkende proteiner (SLP) i peroksisomlumen
<ol><li>Frø de ønskede cellene på glassbunnede cellekulturretter. For eksperimentet her, frø 2 x 105 humane SHSY5Y-celler i 840 μL kulturmedium (DMEM / F-12 supplert med 10% føtal bovint serum og 1% penicillin / streptomycin) eller 6 x 104 mus NIH3T3 celler i 840 μL kulturmedium (DMEM supplert med 10% bovint serum og 1% penicillin / streptomycin) på en 35 mm kulturskål...

<ol>
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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. 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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>

Denne protokollen beskriver hvordan man utløser Stub1-mediert pexophagy i cellekulturer ved å heve peroksisomale ROS-nivåer med lys. Siden protokollen er avhengig av fargeassistert ROS-generering, må man sikre tilstrekkelig uttrykk for diKillerRed-VKSKL eller fargemerket SLP-ligandfarging i cellene av interesse. Gitt at forskjellige celletyper eller celler med forskjellig genetisk bakgrunn kan ha peroksisomer med litt forskjellige egenskaper, må man kanskje justere de nøyaktige belysningsforholdene for å sikre ind...

Peroksisomer er enkeltmembranbundne organeller tilstede i de fleste eukaryote celler. Peroksisomer er et metabolsk rom som er essensielt for å utføre beta-oksidasjon av svært langkjedede fettsyrer, purinkatabolisme og eterfosfolipid og gallesyresyntese1. Peroksisomavledet acetyl-CoA kontrollerer lipidhomeostase ved å regulere den sentrale signaleringen i metabolisme2. Derfor er det ingen overraskelse at kompromitterte peroksisomale funksjoner er underforstått i ulike sykdommer, inkludert nevrodegenerative lidelser, aldring, kreft, fedme og diabetes 3,4,5. En viktig prosess i vedlikehold av peroksisomal drift er pexophagy. Pexophagy er en katabolsk prosess for selektiv omsetning av peroksisomer ved autofagi. Celler bruker pexophagy for å kontrollere mengden og kvaliteten på peroksisomer, og dermed sikre riktig peroksisomalfunksjon. En nylig studie viste at peroksisomtap forårsaket av mutasjoner i PEX1 peroksisomal biogenesefaktor 1 og 6 skyldes ukontrollert pexophagy6. Spesielt har 65% av alle pasienter med peroksisombiogeneseforstyrrelse (PBD) mangler i det peroksisomale AAA ATPase-komplekset, sammensatt av PEX1, PEX6 og PEX26 i pattedyrceller7.
En rekke metoder kan brukes til å initiere og studere pexophagy. I gjær utløses peksofagi når de tilførte næringsstoffene byttes fra peroksisomavhengige karbonkilder til peroksisomuavhengige karbonkilder (til lavere cellulære peroksisomtall)8. For eksempel induserer overføringen av metanoldyrkede Pichia pastoris-celler fra metanolmedium til glukosemedium og etanolmedium mikropexophagy og makropexophagy, henholdsvis 8,9,10. Micropexophagy sekvestrere grupperte peroksisomer for nedbrytning ved å omforme vakuolen for å danne kopplignende vakuolære sekvestreringsmembraner og en lokklignende struktur kalt mikropexophagy-spesifikk membranapparat (MIPA). I makropexophagy blir individuelle peroksisomer oppslukt av dobbeltmembranstrukturer kjent som pexophagosomes, etterfulgt av fusjon med vakuolen for nedbrytning 8,9,10. Fosforyleringen av peksofagireseptorer, som Atg36p i Saccharomyces cerevisiae og Atg30p i Pichia pastoris, er avgjørende for reseptorene for å rekruttere kjerneautofagimaskiner og for å lette peroksisomal målretting mot autofagosomer 8,11.
I pattedyrceller kan pexophagy induseres av ubiquitinering. Merking av peroksisomale membranproteiner PMP34 eller PEX3 med ubiquitin på cytosolisk side induserer pexophagy12. Overekspresjon av PEX3 induserer peroksisom-ubiquitinering og peroksisomeliminasjon av lysosomer13. I tillegg svekker fusjonen av PEX5 med en C-terminal EGFP eksporten av monoubiquitinert PEX5 og resulterer i pexophagy14. På den annen side kan pexophagy også utløses av H 2 O2 behandling. Peroksisomer produserer reaktive oksygenarter (ROS); Spesielt produserer det peroksisomale enzymet Acox1, som katalyserer det første trinnet med beta-oksidasjon av svært langkjedede fettsyrer (> 22 karbon), ikke bare acetyl-CoA, men også peroksisomal ROS. Som svar på de økte ROS-nivåene under H 2 O2-behandling, aktiverer pattedyrceller pexophagy for å senke ROS-produksjonen og lindre stress. Det har blitt rapportert at H 2O2 -behandling driver rekruttering av ataksi-telangiektasi mutert (ATM) til peroksisomer. ATM fosforylerer deretter PEX5 for å fremme peroksisomal omsetning av pexophagy15.
Siden peroksisomer er ROS-genererende sentre, er de også utsatt for ROS-skade. ROS-fremkalte peroksisomale skader tvinger celler til å aktivere pexophagy for å initiere peroksisomkvalitetskontrollveier (fjerning av det skadede peroksisomet ved autofagi). Her skisserer vi en tilnærming for on-demand utløsing av ROS-fremkalt peroksisomal skade. Protokollen utnytter lysaktivert ROS-produksjon innenfor organeller 16,17,18,19,20 (figur 1). Fargemerkede peroksisomer belyses, noe som fører til ROS-produksjon i peroksisomale lumen, som spesifikt utløser peroksisomalskade. Ved hjelp av denne protokollen er det vist at ROS-stressede peroksisomer fjernes gjennom en ubiquitinavhengig nedbrytningsvei. ROS-stressede peroksisomer rekrutterer ubiquitin E3-ligasen Stub1 for å tillate deres oppslukt i autofagosomer for individuell fjerning av pexophagy16. Man kan bruke denne protokollen til å sammenligne skjebnen til skadde og friske peroksisomer i samme celle ved time-lapse-mikroskopi. Metoden kan også brukes til å skade alle peroksisomer (i alle celler) globalt på en kulturskål, noe som muliggjør den biokjemiske analysen av pexofagy-banen.

<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

Pattedyrceller kan snu peroksisomer gjennom Stub1-mediert pexophagy. Banen tillater potensielt cellulær kontroll av mengden og kvaliteten på peroksisomer. Under denne prosessen translokerer varmesjokkprotein 70 og ubiquitin E3-ligasen, Stub1, på peroksisomer som skal overføres for å initiere pexophagy. Stub1-ligaseaktiviteten tillater akkumulering av ubiquitin og andre autofagirelaterte moduler på målrettede peroksisomer. Forhøyede nivåer av reaktive oksygenarter (ROS) i peroksisomale lumen kan aktivere Stub1-mediert pexophagy. Man kan derfor bruke fargeassistert ROS-generering for å utløse og overvåke denne banen. Denne artikkelen skisserer prosedyrene for bruk av to klasser av fargestoffer, fluorescerende proteiner og syntetiske fluoroforer, for å initiere pexophagy i pattedyrcellekulturer. Disse fargeassisterte ROS-generasjonsbaserte protokollene kan ikke bare brukes til å målrette alle peroksisomer i en cellepopulasjon globalt, men kan også tillate manipulering av individuelle peroksisomer i enkeltceller. Vi beskriver også hvordan Stub1-mediert pexophagy kan følges ved hjelp av levende cellemikroskopi.

Stub1-mediert pexophagy induksjonsskjema vist her utnytter fargeassistert ROS-generering innenfor peroksisomlumen. Denne operasjonen krever minimal lysintensitet. Peroksisomer som inneholder fluorescerende proteiner eller fargestoffer kan derfor belyses ved hjelp av standard laserskanning konfokale mikroskoper. Fokal belysning fører til øyeblikkelig og lokalisert ROS-produksjon innenfor individuelle peroksisomer, som indikert av den fluorescerende reporteren roGFP2-VKSKL (figur 9). Vi avb...

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

Monitoring Stub1-Mediated Pexophagy

Denne protokollen gir instruksjoner for å utløse og overvåke Stub1-mediert pexophagy i levende celler.

Overvåking av stubb1-mediert pexophagy

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

Peroksisomer utfører beta-oksidasjon og er utsatt for ROS-skade. Denne metoden gjør det mulig å undersøke hvordan celler håndterer ROS-stressede peroksisomer. Denne protokollen brukes ikke bare til å globalt målrette alle peroksisomer i en cellepopulasjon, men kan også tillate manipulering av de enkelte peroksisomer i enkeltceller.Fargeassistert ROS-generasjon kan også brukes til å målrette organeller utenfor peroksisomer for å undersøke hvordan celler håndterer organelle skader. For å begynne, frø to ganger 10 til den femte menneskelige SHSY5Y-celler eller seks ganger 10 til den fjerde musen NIH H3T3-celler i 840 mikroliter kulturmedium på glassbunnede 35-millimeter kulturretter med en 20 millimeter diameter glassmikrobrønn. Dyrk cellene under 5% karbondioksid ved 37 grader Celsius i 24 timer.Deretter transfekter du cellene med ønskede plasmider ved hjelp av et transfeksjonsreagens eller SHSY5Y-celletransfeksjon. Fortynn plasmider i 55 mikroliter serumfri DMEM / F-12 og fortynn en mikroliter transfeksjonsreagens i 55 mikroliter serumfri DMEM / F-12. Kombiner det fortynnede DNA med det fortynnede reagenset.Bland forsiktig og inkuber i 30 minutter ved romtemperatur. Legg komplekset til 20 millimeter mikrobrønn som tidligere er belagt med 100 mikroliter kulturmedium som inneholder SHSY5Y-celler uten antibiotika. Rist fatet forsiktig frem og tilbake.Utfør NIH 3T3 celletransfeksjon som vist før, men erstatt redusert serummedium i stedet for serumfritt DMEM / F-12 for plasmidfortynning og transfeksjonsreagens. Etter to timers transfeksjon, fjern transfeksjonsreagensholdig medium og tilsett en milliliter friskt kulturmedium. Inkuber NIH 3T3- eller SHSY5Y-cellene ved 37 grader Celsius og 5% karbondioksid i 24 timer.For lysaktivert ROS-produksjon, 24 timer etter transfeksjon, fjern kulturmediet og tilsett 10 mikroliter 200 nanomolar HaloTag TMR-ligand eller 200 nanomolar Janelia Fluor 646 HaloTag-ligand på 20 millimeter glassmikrobrønn. Overfør parabolen til en 37 grader Celsius og 5% karbondioksidinkubator og la den stå i en time for å flekke cellene. Etter en time, fjern fargemediet grundig og tilsett en milliliter friskt kulturmedium.Plasser en glassbunnet tallerken som inneholder de ønskede cellene på et laserskanning konfokalmikroskop utstyrt med en scene-topp inkubator. I programvaren klikker du på Verktøyvindu, velger Ocular, og klikker på DIA-knappen for å slå på det overførte lyset. Se parabolen gjennom mikroskopetokularet og bring cellene i fokus ved å vri fokusknappen.Velg LSM og klikk på Dye Detector Select-knappen. Dobbeltklikk på de ønskede fargestoffene i popup-vinduet for å velge passende laser- og filterinnstillinger for avbildning av cellene. Klikk på Live4x i Live-panelet for å starte rask laserskanning for å starte screening for fluorescerende signaler fra cellene.Flytt scenen rundt ved hjelp av joystick-kontrolleren og finn mediet diKillerRed VKSKL eller SLP-VKSKL-uttrykkende celler for å påføre ROS-stress på peroksisomer. Skaff deg et bilde av ønsket celle. Klikk på Verktøyvindu og velg LSM-stimulering.Klikk deretter på ellipseknappen og bruk musen i Live Image-vinduet til å tegne en avkastning på 15 mikrometer i diameter som inneholder de ønskede peroksisomer som ROS-stress vil bli påført. For å påføre ROS-stress, bruk 561 nanometer for celler med diKillerRed VKSKL eller TMR-merket SLP-ligand og bruk 640 nanometer for celler farget med Janelia Fluor 646-merket SLP-ligand. Velg laserbølgelengden for påføring av ROS-spenningen.Angi ønsket laserprosent og varighet. For ROS-produksjon i peroksisomer, klikk på Oppkjøp og deretter Stimulering-knappen for å starte skanningen med 561 eller 640 nanometer lys gjennom avkastningen i 30 sekunder hver. Denne stimuleringen tilsvarer omtrent 5% lasereffektinnstillinger for 561 og 640 nanometer.Etter 30 sekunder med laserbelysning mister peroksisomer innenfor avkastningen sine diKillerRed VKSKL-, TMR- eller Janelia Fluor 646-signaler. Dette signaltapet gjør det mulig å skille de opplyste og ikke-opplyste peroksisomer i cellen. For å overvåke pexophagean levende celler, følg translokasjonen av EGFP-merket Stub1, Hsp70, ubiquitin, p62 eller LC3B på de opplyste eller ROS-stressede peroksisomer ved tidsforløpsavbildning ved hjelp av 10-minutters intervaller mellom rammer.Fokal belysning fører til øyeblikkelig og lokalisert ROS-produksjon innenfor individuelle peroksisomer som indikert av den fluorescerende reporteren roGFP2-VKSKL. DiKillerRed VKSKL-bildet ble tatt etter at alle roGFP2-VKSKL-målingene ble gjort for å indikere de skadede peroksisomenes plassering. Dataene viser også at ubelyste peroksisomer ikke påvirkes, noe som indikerer metodikkens presisjon.Denne metodikken ble brukt til å overvåke Stub1-mediert pexophagy. Under denne prosessen vises Hsp70, Stub1, ubiquitinerte proteiner, autofagiadaptere p62 og LC3B senere i ROS-stressede peroksisomer for å drive pexophagy. Ved å bruke samme strategi var det mulig å samtidig skade alle peroksisomer på en kulturskål for biokjemisk karakterisering av Stub1-mediert pexophagy.Før LED-belysning var EGFP-ubiquitinfluorescens homogen i cytoplasma og kolokaliserte ikke med peroksisomer. Etter ni timer med LED-belysning akkumulerte EGFP-ubiquitin-signalene på alle peroksisomer i cellene som vokste i en konfokal tallerken. Ved hjelp av denne protokollen fant vi at ROS-stressede peroksisomer fjernes gjennom en ubiquitinavhengig nedbrytningsvei.ROS-stressede peroksisomer rekrutterer ubiquitin E3 ligase Stub1 for å tillate fjerning av dem ved pexophagy.

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

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.

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.

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.

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.

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.

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.

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.

Langsiktig Dødelig toksisitet Test med krepsdyret 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.

Sammen Analyser for Monitoring Protein Refolding i<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.

Valgt Reaction Monitoring massespektrometri for Absolute Protein Kvantifisering

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