Name: methods to study mrp4-containing macromolecular complexes in the regulation of fibroblast migration
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Description: Watch this Scientific Journal Video about Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration at JoVE.com

This work was supported by National Institutes of Health grants R01-DK080834 and R01-DK093045. We thank J. Denise Wetzel, CCHMC Medical Writer, for editing of the manuscript.

1. Ingenuity Pathway Analysis <ol><li> Upload Protein-interactome Datasæt <ol><li> Sæt proteiner / gener af interesse i et regneark med deres unikke gen-id&#39;er (helst gen symboler og gen-identifikationsnumre som opnået med massespektrometriske data). </li><li> Tildel en kolonne i regnearket for Gene id-nummer og en kolonne for observationelle værdi (f.eks., Fold-ændring eller p-værdi). Vælg &quot;indeholder kolonneoverskriften &#39;mulighed for at se kolonneoverskrifterne. </li><li> Upload datasætt...

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Cell migration is an intricate process that plays indispensable roles in many important physiological events including wound healing1,2. Aberrant cell migrations may cause catastrophic events, such as tumor metastasis and angiogenesis24,25. Therefore, fine-tuned regulation of cell migration is required to maintain normal body function.
Using high-content microscopy18, we demonstrated that MRP4-deficient MEFs migrate faster compared to wild-type fibroblasts...

Cell migration er en kompliceret multi-trins proces. Undersøgelser har vist, at under migration celler polariseret i for- og bagkanten. Ved at følge den ekstracellulære matrix, den førende kant giver trækkraft er nødvendig for cellen kroppen at bevæge sig fremad. Endelig efterstillede kant udgivelser bag vedhæftede filer og fuldender migration cyklus 1,2. Cell polarisering til effektiv cellemigration reguleres af rumlig adskillelse af intracellulær signalering. Cellular second messengers, såsom cAMP, mægle opdeling af signalering begivenheder, der kræves for finjusteret retningsbestemt celle migration 3,4. Differentieret ophobninger af cAMP og cAMP-afhængig kinase PKA aktivitet på forkant spiller nøgleroller i retningsbestemt cellemigration 5,6. Ved phosphorylering små GTPaser såsom Ras-relateret C3 botulinustoksin substrat (Rac) og celledeling kontrolprotein 42 homolog eller Cdc42, PKA aktiverer actin-relateret protein 2/3 (Arp 2/3) ved forkanten og inducerer dannelsen af lamellipodia 7-9. PKA phosphorylerer også en anti-capping agent, vasodilator stimuleret phosphoprotein (VASP), derved regulerer oscillerende cyklusser af membran udvidelse og tilbagetrækning 10,11. I celler, der cAMP-niveauer reguleres af tre hovedprocesser: i) syntese ved adenylatcyclase, ii) nedbrydning af phosphodiesteraser, og iii) transport af membranbundne effluxtransportører 3. Multilægemiddelresistens protein 4 (MRP4), et medlem af ATP-bindende kassette (ABC) familie af membrantransportører, funktioner som en endogen efflukstransporteren af ​​cykliske nukleotider. Derfor kan MRP4 regulere intracellulære cAMP-niveauer og cAMP-afhængig cellulær signalering 11-13. Vi har tidligere vist, at i Mrp4 - / - fibroblaster indeholder relativt højere niveauer af cykliske nukleotider og migrere hurtigere compared til Mrp4 + / + fibroblaster 14. Vi rapporteret også en bifasisk effekt af cykliske nukleotider på fibroblast migration. Baseret på tidligere studier og vores fund, at Mrp4 - / - fibroblaster indeholder mere polariseret cAMP i løbet af migration, vi hypotese, at dette MRP4-medieret regulering af fibroblast migration er cAMP-afhængig. For at forstå den nedstrøms mekanisme, tog vi en direkte alligevel flerstrenget tilgang. For at identificere de proteiner, der er forbundet og i samspil med MRP4, vi immunopræcipiterede MRP4-holdige makromolekylære komplekser fra HEK293 celler at over udtrykker MRP4. Brug massespektrometri, identificerede vi flere MRP4-interagerende proteiner og analyseret deres sammenkobling hjælp Ingenuity Pathway Analysis (IPA). IPA er et nyttigt værktøj til at analysere protein-protein interaktioner (både strukturelle og funktionelle) og udforske deres bidrag, især fysiologiske og patologiskebegivenheder baseret på litteraturen og eksperimentelle beviser 15,16. IPA indikerede, at F-actin er et stort nedstrømsmål af MRP4 i forbindelse med cellemigrering hvor cAMP og cGMP er nøglen signalmolekyler 17. Disse data blev yderligere bekræftet ved højinformativ mikroskopi. High-indhold mikroskopi kan fange og analysere celle adfærd, såsom celle migration i en mere bekvem, nøjagtig og high-throughput måde 18. De høje-indhold mikroskopi data viste, at virkningen af MRP4 på fibroblast migration er helt afskaffes ved afbrydelse af actincytoskelettet eller inhibering af PKA 17. Desuden har vi brugt en Forster resonans (FRET) -baserede PKA sensor til at overvåge PKA dynamik i migrerende celler i realtid. FRET-baserede kinase sensorer består normalt af specifikke phosphoryleringssubstrat peptider flankeret af den fælles fiskeripolitik og YFP fluoroforer 19-21. pmAKAR3 er en forbedret og migmbrane målrettet FRET-baserede PKA sensor, der indeholder forkhead-associerede domæne 1 (FHA1) og PKA substrat sekvens LRRATLVD 5,22. Phosphorylering af pmAKAR3 af PKA katalytiske subunit stigninger FRET signal mellem FFP og YFP 19. Indsættelse af et lipid modifikation domæne ind i sensoren målretter det til plasmamembranen til overvågning PKA dynamik, specielt mod membranen kammer 23. Brug af pmAKAR3, demonstrerede vi at forkanten migrere Mrp4 - / - fibroblaster udviste mere polariseret PKA aktivitet end Mrp4 + / + fibroblaster, som igen udbyggede kortikale actin formationen ved cellens førende kant 17. Tilsammen udgør disse begivenheder resulterede i bedre cellulær polarisering og hurtigere retningsbestemt celle migration i fravær af MRP4. Vores specifikke og direkte tilgang identificeret vigtige downstream mål for MRP4 og giver en vigtig, men somendnu uudforskede mekanisme til MRP4-afhængig regulering af fibroblast migration.

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methods to study mrp4-containing macromolecular complexes in the regulation of fibroblast migration

Multilægemiddelresistens protein 4 (MRP4) er et medlem af ATP-bindende kassette-familien af ​​membrantransportører og er en endogen efflukstransporteren af ​​cykliske nukleotider. Ved at modulere intracellulær cyklisk nukleotid koncentration, kan MRP4 regulere flere cykliske nukleotid-afhængige cellulære begivenheder, herunder celle migration. Tidligere påviste vi, at i fravær af MRP4, fibroblastceller indeholder højere niveauer af intracellulære cykliske nukleotider og kan migrere hurtigere. For at forstå de underliggende mekanismer af dette fund, vedtog vi en direkte alligevel flerstrenget tilgang. Først, vi isoleret potentielle interagerende protein komplekser af MRP4 fra en MRP4 overekspression celle system ved hjælp af immunpræcipitation efterfulgt af massespektrometri. Efter identifikation unikke proteiner i MRP4 interactome, vi udnyttet Ingenuity Pathway Analysis (IPA) for at undersøge den rolle, disse protein-protein interaktioner i forbindelse med signaltransduktion. Vi belyst den potiale rolle MRP4 proteinkompleks i cellemigrering og identificeret F-actin som en vigtig mediator af virkningen af ​​MRP4 på cellemigrering. Denne undersøgelse understregede også betydningen af ​​cAMP og cGMP som centrale aktører i de vandrende fænomener. Ved hjælp af høj-indhold mikroskopi, udførte vi celle-migration assays og observerede, at virkningen af ​​MRP4 på fibroblast migration er helt afskaffet ved afbrydelse af actincytoskelettet eller inhibering af cAMP-afhængig kinase A (PKA). At visualisere signalering modulationer i en vandrende celle i realtid, vi udnyttet en FRET-baseret sensor til måling PKA aktivitet og fandt, at tilstedeværelsen af mere polariseret PKA aktivitet nær forkanten af migrerende Mrp4 - / - fibroblast, sammenlignet med Mrp4 + / + fibroblaster. Dette vil til gengæld øget kortikal actin dannelse og augmented processen med migration. Vores tilgang gør det muligt at identificere de proteiner, der virker nedstrøms til MRP4 og giver os en overbetragtning af den involveret i MRP4-afhængig regulering af fibroblast migration mekanisme.

For at undersøge virkningen af MRP4 på fibroblast migration, anvendte vi en sårhelende assay under anvendelse højinformativ mikroskopi 14. Præcise sår blev foretaget på sammenflydende monolag af MEF&#39;er isoleret fra enten Mrp4 - / - eller Mrp4 + / + mus, og billeder blev taget ved 1 intervaller time i 24 timer. Vi observerede en højere vandringshastighed for Mrp4 - / - MEF&#39;er ...

Watch this Scientific Journal Video about Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration at JoVE.com

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

MRP4 regulerer forskellige cykliske nukleotid-afhængig signalering arrangementer, herunder en nylig belyst rolle i celle migration. Vi beskriver en direkte, men flerstrenget tilgang at optrævle de nedstrøms molekylære mål for MRP4 resulterer i identifikation af et unikt MRP4 interactome der spiller centrale roller i finjusteret regulering af fibroblast migration.

Metoder til at studere Mrp4-holdige Makromolekylær Komplekser i forordningen af ​​fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

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Metoder til at studere Mrp4-holdige Makromolekylær Komplekser i forordningen af ​​fibroblast Migration

Metoder til at studere Mrp4-holdige Makromolekylær Komplekser i forordningen af fibroblast Migration