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

Detta arbete stöddes delvis av ett MOST 111-2311-B-001-019-MY3 forskningsbidrag från National Science and Technology Council i Taiwan.

1. Beredning av celler som uttrycker diKillerRed eller självmärkande proteiner (SLP) i peroxisomlumen
<ol><li>Frö de önskade cellerna på glasbottnade cellodlingsrätter. För experimentet här, frö 2 x 105 humana SHSY5Y-celler i 840 μL odlingsmedium (DMEM / F-12 kompletterat med 10% fetalt bovint serum och 1% penicillin / streptomycin) eller 6 x 104 mus NIH3T3-celler i 840 μL odlingsmedium (DMEM kompletterat med 10% bovint serum och 1% penicillin / streptomycin) på en 35 ...

<ol>
	<li>Wanders, R. J., Waterham, H. R., Ferdinandusse, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+interplay+between+peroxisomes+and+other+subcellular+organelles+including+mitochondria+and+the+endoplasmic+reticulum.">Metabolic interplay between peroxisomes and other subcellular organelles including mitochondria and the endoplasmic reticulum.</a> Frontiers in Cell and Developmental Biology. 3, 83 (2016).</li><li>He, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetyl-CoA+derived+from+hepatic+peroxisomal+&#946;-oxidation+inhibits+autophagy+and+promotes+steatosis+via+mTORC1+activation.">Acetyl-CoA derived from hepatic peroxisomal &#946;-oxidation inhibits autophagy and promotes steatosis via mTORC1 activation.</a> Molecular Cell. 79 (1), 30.e4-42.e4 (2020).</li><li>Cipolla, C. 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>

Detta protokoll beskriver hur man utlöser Stub1-medierad pexofagi inom cellkulturer genom att höja peroxisomala ROS-nivåer med ljus. Eftersom protokollet bygger på färgassisterad ROS-generering måste man säkerställa tillräckligt uttryck av diKillerRed-VKSKL eller färgämnesmärkt SLP-ligandfärgning i cellerna av intresse. Med tanke på att olika celltyper eller celler med olika genetisk bakgrund kan hysa peroxisomer med något olika egenskaper, kan man behöva ställa in de exakta belysningsförhållandena fö...

Peroxisomer är enkelmembranbundna organeller som finns i de flesta eukaryota celler. Peroxisomer är ett metaboliskt fack som är nödvändigt för att utföra beta-oxidation av mycket långkedjiga fettsyror, purinkatabolism och eterfosfolipid- och gallsyrasyntes1. Peroxisom-härledd acetyl-CoA kontrollerar lipidhomeostas genom att reglera den centrala signaleringen i metabolism2. Därför är det ingen överraskning att komprometterade peroxisomala funktioner är underförstådda i olika sjukdomar, inklusive neurodegenerativa störningar, åldrande, cancer, fetma och diabetes 3,4,5. En viktig process vid upprätthållandet av peroxisomal drift är pexofagi. Pexophagy är en katabol process för selektiv omsättning av peroxisomer genom autofagi. Celler använder pexophagy för att hjälpa till att kontrollera kvantiteten och kvaliteten på peroxisomer, vilket säkerställer korrekt peroxisomal funktion. En nyligen genomförd studie visade att peroxisomförlust orsakad av mutationer i PEX1 peroxisomal biogenes faktor 1 och 6 är resultatet av okontrollerad pexofagi6. I synnerhet har 65% av alla patienter med peroxisombiogenessjukdom (PBD) brister i det peroxisomala AAA ATPas-komplexet, bestående av PEX1, PEX6 och PEX26 i däggdjursceller7.
Ett antal metoder kan användas för att initiera och studera pexofagi. I jäst utlöses pexophagy när de tillförda näringsämnena byts från peroxisomberoende kolkällor till peroxisomoberoende kolkällor (för att sänka cellulära peroxisomantal)8. Exempelvis inducerar överföringen av metanolodlade Pichia pastoris-celler från metanolmedium till glukosmedium och etanolmedium mikropexofagi respektive makropexofagi 8,9,10. Micropexophagy sekvestrerar grupperade peroxisomer för nedbrytning genom att omforma vakuolen för att bilda koppliknande vakuolära sekvestreringsmembran och en lockliknande struktur som kallas mikropexophagy-specifik membranapparat (MIPA). I makropexofagi uppslukas enskilda peroxisomer av dubbelmembranstrukturer som kallas pexofagosomer, följt av fusion med vakuolen för nedbrytning 8,9,10. Fosforyleringen av pexofagireceptorer, såsom Atg36p i Saccharomyces cerevisiae och Atg30p i Pichia pastoris, är avgörande för receptorerna att rekrytera kärnautofagimaskiner och för att underlätta peroxisomal inriktning mot autofagosomer 8,11.
I däggdjursceller kan pexofagi induceras genom ubiquitination. Märkning av de peroxisomala membranproteinerna PMP34 eller PEX3 med ubiquitin på den cytosoliska sidan inducerar pexofagi12. Överuttryck av PEX3 inducerar peroxisomubiquitinering och peroxisomeliminering av lysosomer13. Dessutom försämrar fusionen av PEX5 med en C-terminal EGFP exporten av monoubiquitinerad PEX5 och resulterar i pexophagy14. Å andra sidan kan pexophagy också utlösas avH2O2-behandling. Peroxisomer producerar reaktiva syreradikaler (ROS); specifikt producerar det peroxisomala enzymet Acox1, som katalyserar det första steget av beta-oxidation av mycket långkedjiga fettsyror (> 22 kol), inte bara acetyl-CoA utan också peroxisomal ROS. Som svar på de ökade ROS-nivåerna underH2O2-behandlingaktiverar däggdjursceller pexofagi för att sänka ROS-produktionen och lindra stress. Det har rapporterats attH2O2-behandlingdriver rekryteringen av ataxi-telangiektasi muterad (ATM) till peroxisomer. ATM fosforylerar sedan PEX5 för att främja peroxisomal omsättning genom pexophagy15.
Eftersom peroxisomer är ROS-genererande centra är de också benägna att ROS-skador. ROS-framkallade peroxisomala skador tvingar celler att aktivera pexofagi för att initiera peroxisomkvalitetskontrollvägar (avlägsnande av den skadade peroxisomen genom autofagi). Här beskriver vi ett tillvägagångssätt för on-demand utlösning av ROS-framkallad peroxisomal skada. Protokollet utnyttjar ljusaktiverad ROS-produktion inom organellerna 16,17,18,19,20 (figur 1). Färgmärkta peroxisomer belyses, vilket leder till ROS-produktion inom peroxisomalt lumen, vilket specifikt utlöser peroxisomal skada. Med hjälp av detta protokoll visas att ROS-stressade peroxisomer avlägsnas genom en ubiquitinberoende nedbrytningsväg. ROS-stressade peroxisomer rekryterar ubiquitin E3-ligaset Stub1 för att tillåta deras uppslukning i autofagosomer för individuellt avlägsnande av pexophagy16. Man kan använda detta protokoll för att jämföra ödet för skadade och friska peroxisomer inom samma cell genom time-lapse-mikroskopi. Metoden kan också användas för att globalt skada alla peroxisomer (i alla celler) på en odlingsskål, vilket möjliggör biokemisk analys av pexofagivägen.

<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

Däggdjursceller kan vända peroxisomer genom Stub1-medierad pexofagi. Vägen tillåter potentiellt cellulär kontroll av kvantiteten och kvaliteten på peroxisomer. Under denna process translokerar värmechockprotein 70 och ubiquitin E3-ligaset, Stub1, till peroxisomer som ska vändas för att initiera pexofagi. Stub1-ligasaktiviteten möjliggör ackumulering av ubiquitin och andra autofagirelaterade moduler på riktade peroxisomer. Förhöjning av nivåerna av reaktiva syreradikaler (ROS) inom peroxisomalt lumen kan aktivera Stub1-medierad pexofagi. Man kan därför använda färgämnesassisterad ROS-generering för att utlösa och övervaka denna väg. Denna artikel beskriver procedurerna för användning av två klasser av färgämnen, fluorescerande proteiner och syntetiska fluoroforer, för att initiera pexofagi inom däggdjurscellkulturer. Dessa färgämnesassisterade ROS-generationsbaserade protokoll kan inte bara användas för att rikta in sig på alla peroxisomer inom en cellpopulation globalt utan kan också möjliggöra manipulation av enskilda peroxisomer i enskilda celler. Vi beskriver också hur Stub1-medierad pexofagi kan följas med hjälp av levande cellmikroskopi.

Det Stub1-medierade pexophagy induktionsschemat som visas här utnyttjar färgassisterad ROS-generering inom peroxisomlumen. Denna operation kräver minimala ljusintensiteter. Peroxisomer som innehåller fluorescerande proteiner eller färgämnen kan därför belysas med hjälp av vanliga laserscanningskonfokalmikroskop. Fokal belysning leder till momentan och lokal ROS-produktion inom enskilda peroxisomer, vilket indikeras av den fluorescerande reportern roGFP2-VKSKL (figur 9). Vi avbildad...

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

Monitoring Stub1-Mediated Pexophagy

Detta protokoll innehåller instruktioner för utlösning och övervakning av Stub1-medierad pexofagi i levande celler.

Övervakning av Stub1-medierad pexofagi

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

Peroxisomer utför betaoxidation och är benägna att ROS-skador. Denna metod gör det möjligt att undersöka hur celler hanterar ROS-stressade peroxisomer. Detta protokoll används inte bara för att globalt rikta in sig på alla peroxisomer inom en cellpopulation utan kan också tillåta manipulation av de enskilda peroxisomerna i enskilda celler.Färgassisterad ROS-generering kan också användas för att rikta organeller utanför peroxisomer för att undersöka hur celler hanterar organellskador. Till att börja med, frö två gånger 10 till de femte mänskliga SHSY5Y-cellerna eller sex gånger 10 till den fjärde musen NIH H3T3-celler i 840 mikroliter odlingsmedium på glasbottnade 35-millimeter odlingsskålar med en 20 millimeter diameter glasmikrobrunn. Odla cellerna under 5% koldioxid vid 37 grader Celsius i 24 timmar.Transfektera sedan cellerna med önskade plasmider med hjälp av ett transfektionsreagens eller SHSY5Y-celltransfektion. Späd plasmiderna i 55 mikroliter serumfritt DMEM/F-12 och späd en mikroliter transfektionsreagens i 55 mikroliter serumfritt DMEM/F-12. Kombinera det utspädda DNA:t med det utspädda reagenset.Blanda försiktigt och inkubera i 30 minuter vid rumstemperatur. Lägg komplexet till 20-millimeters mikrobrunnen som tidigare pläterats med 100 mikroliter odlingsmedium innehållande SHSY5Y-celler utan antibiotika. Skaka försiktigt skålen fram och tillbaka.Utför NIH 3T3-celltransfektion som visats tidigare men ersätt reducerat serummedium istället för serumfritt DMEM / F-12 för plasmidutspädning och transfektionsreagens. Efter två timmars transfektion, avlägsna det transfektionsreagensinnehållande mediet och tillsätt en milliliter färskt odlingsmedium. Inkubera NIH 3T3- eller SHSY5Y-cellerna vid 37 grader Celsius och 5% koldioxid i 24 timmar.För ljusaktiverad ROS-produktion, vid 24 timmar efter transfektion, avlägsna odlingsmediet och tillsätt 10 mikroliter 200 nanomolar HaloTag TMR-ligand eller 200 nanomolar Janelia Fluor 646 HaloTag-ligand på 20-millimeters glasmikrobrunnen. Överför skålen till en inkubator på 37 grader Celsius och 5% koldioxid och låt den stå i en timme för att färga cellerna. Efter en timme, ta bort färgningsmediet noggrant och tillsätt en milliliter färskt odlingsmedium.Placera en glasbottnad skål som innehåller de önskade cellerna på ett laserskanningskonfokalmikroskop utrustat med en sceninkubator. Klicka på Verktygsfönster i programvaran, välj Okulär, och klicka på DIA-knappen för att slå på det överförda ljuset. Visa skålen genom mikroskopokularet och sätt cellerna i fokus genom att vrida på fokusratten.Välj LSM och klicka på knappen Välj färgdetektor. Dubbelklicka på önskade färgämnen i popup-fönstret för att välja lämpliga laser- och filterinställningar för avbildning av cellerna. Klicka på Live4x i Live-panelen för att starta snabb laserskanning för att börja screena för cellernas fluorescerande signaler.Flytta runt scenen med joystickkontrollen och lokalisera mediet diKillerRed VKSKL eller SLP-VKSKL-uttryckande celler för att applicera ROS-stress på peroxisomerna. Skaffa en bild av önskad cell. Klicka på Verktygsfönster och välj LSM-stimulering.Klicka sedan på ellipsknappen och använd musen i Live Image-fönstret för att rita en ROI på 15 mikrometer i diameter som innehåller de önskade peroxisomerna som ROS-spänning kommer att appliceras på. För att applicera ROS-stress, använd 561 nanometer för celler med diKillerRed VKSKL eller TMR-märkt SLP-ligand och använd 640 nanometer för celler färgade med Janelia Fluor 646-märkt SLP-ligand. Välj laservåglängden för applicering av ROS-spänningen.Ange önskad laserprocent och varaktighet. För ROS-produktion i peroxisomerna, klicka på Skaffa och sedan stimuleringsknappen för att initiera skanningen med 561 eller 640 nanometer ljus genom ROI i 30 sekunder vardera. Denna stimulering motsvarar cirka 5% lasereffektinställningar för 561 och 640 nanometer.Efter 30 sekunders laserbelysning förlorar peroxisomer inom ROI sina diKillerRed VKSKL-, TMR- eller Janelia Fluor 646-signaler. Denna signalförlust gör det möjligt att skilja de upplysta och icke-upplysta peroxisomerna i cellen. För att övervaka de pexophagean levande cellerna, följ translokationen av EGFP-märkta Stub1, Hsp70, ubiquitin, p62 eller LC3B till de upplysta eller ROS-stressade peroxisomerna genom tidsfördröjningsavbildning med 10-minuters intervall mellan ramar.Fokal belysning leder till momentan och lokal ROS-produktion inom enskilda peroxisomer som indikeras av den fluorescerande reportern roGFP2-VKSKL. DiKillerRed VKSKL-bilden togs efter att alla roGFP2-VKSKL-mätningar gjordes för att indikera de skadade peroxisomernas plats. Data visar också att obelysta peroxisomer inte påverkas, vilket indikerar metodens precision.Denna metod användes för att övervaka Stub1-medierad pexofagi. Under denna process uppträder Hsp70, Stub1, ubiquitinerade proteiner, autofagiadaptrar p62 och LC3B därefter i ROS-stressade peroxisomer för att driva pexofagi. Med samma strategi var det möjligt att samtidigt skada alla peroxisomer på en odlingsskål för den biokemiska karakteriseringen av Stub1-medierad pexofagi.Före LED-belysning, EGFP-ubiquitinfluorescens var homogen i cytoplasman och samlokaliserade inte med peroxisomerna. Efter nio timmars LED-belysning ackumulerades EGFP-ubiquitinsignalerna på alla peroxisomer i cellerna som odlas i en konfokal skål. Med hjälp av detta protokoll fann vi att ROS-stressade peroxisomer avlägsnas genom en ubiquitinberoende nedbrytningsväg.ROS-stressade peroxisomer rekryterar ubiquitin E3-ligasen Stub1 för att möjliggöra borttagning av pexofagi.

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Övervakning Actin demontering med Time-lapse Mikroskopi

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.

Funktionell avbildning med armering, Eyetracking och fysiologiska övervakning

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.

Enda molekyl Metoder för att följa förändringar i tvåskiktsmembran elastiska egenskaper

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.

Övervakning växt hormoner under stressreaktioner

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.

Övervakning Akupunktur Effekter på Human Brain av 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.

Övervakning av hjärtfunktion i Larvernas Drosophila melanogaster För fysiologiska studier

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.

Biologiska testsystem för övervakning Insektsmedel 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.

Långsiktig Dödligt toxicitetstest med Crustacean 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.

Kopplade Analyser för övervakning Proteinåterveckningssteg 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.

Vald Reaction Monitoring masspektrometri för Absolute protein kvantifiering

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