Påvisning af ligand-aktiverede G-protein-koblede receptorer Internalisering af konfokal mikroskopi
Summary
Denne protokol beskriver konfokal mikroskopi påvisning af G-protein-koblet receptor (GPCR) internalisering i pattedyrceller. Det omfatter grundlæggende cellekultur, transfektion, og konfokal mikroskopi procedure og tilvejebringer en effektiv og let fortolkelige metode til at påvise den subcellulære lokalisering og internalisering af fusion-udtrykte GPCR.
Abstract
Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.
Introduction
Celler besidder fragt handel maskiner til at transportere ekstracellulære materialer-såsom ligander, mikroorganismer, næringsstoffer og transmembrane proteiner-ind i cellen for at få information, energi og andre formål 1, 2. Det er afgørende for cellulær homeostase, væv funktion, og den samlede celle overlevelse. Det cellebaserede ekspression af fluorescerende fusionsproteiner er en kraftfuld måde at studere lokaliseringen og internalisering af transmembrane receptorer, såsom G-proteinkoblede receptorer (GPCR), i signalveje 3.
Ekspressionen af fusionsproteiner mærket med grønt fluorescerende protein (GFP) fra goplen Aequorea victoria, kombineret med direkte fluorescens-detektionsteknikker, har vundet bred accept i protein-targeting forskning 4, 5, 6. GFP-baserede detectipå har den fordel, at den tidstro billeddannelse af levende celler samtidig undgå fikseringen artefakter 7. En række alsidige kloningsvektorer er blevet konstrueret til at lette ekspressionen af proteinfusioner til GFP i forskellige celler 8, 9. PEGFP-N1 vektor er en udbredt og kommercialiseret plasmidvektor der koder for et forstærket grønt fluorescerende protein (EGFP), der er optimeret fra vildtype GFP for lysere fluorescens og højere ekspression i mammale celler. At observere det fluorescerende signal af EGFP udtrykkes i pattedyrceller, bør fluorescensmikroskopi udføres med excitation ved 488 nm og emission ved 507 nm, fortrinsvis med konfokal mikroskopi.
Overvågning af subcellulære lokalisering og ligand-medieret internalisering af receptorer er en almindelig teknik til at undersøge signaltransduktion og funktion af GPCR'er 10 </sop>, 11. I de fleste tilfælde, internalisering fører til desensibilisering af GPCR'er (dæmpningen af stimulus respons på trods af tilstedeværelsen af agonister) 12, 13. De internaliserede receptorer kan inkorporeres i lysosomet og nedbrydes, eller de kan recirkuleres tilbage til celleoverfladen, afhængigt af arten af receptorerne og cellelinierne anvendt i forsøgene.
De gonadotropin-frigivende hormon-receptorer (GnRHR) tilhører GPCR-familien og har en typisk GPCR proteinstruktur, med et ekstracellulært aminoterminalt, et intracellulært -COOH terminal, og syv transmembrane (TM) domænerne 14. Transfektion af rekombinant plasmid Sj GnRH-R-EGFP (med GnRH-R-genet fra Sepiella japonica) og konfokal mikroskopi detektion af EGFP-mærkede Sj GnRH-R (udtrykt i HEK293-celler) blev rapporteret previously, og GFP-fluorescens blev etableret som en reporter for transmembran proteinlokalisering assays 15. Nu, internalisering af Sj GnRH-R, aktiveret af S. japonica gonadotropin-frigørende hormon (Sj GnRH), blev demonstreret i tids- og dosisafhængige måder ved konfokal mikroskopi.
Protocol
Representative Results
Discussion
Protokollen præsenteres her tilvejebringer en effektiv og let fortolkelige metode til at påvise den subcellulære lokalisering og internalisering af udtrykt GPCR fusionsprotein i HEK293-celler. Teknikken kan let tilpasses til mange forskellige gener og celletyper, såsom til den cellulære lokalisering og internalisering af corazonin receptoren fra Bombyx mori i HEK293 og BMN celler 16.
Den vellykkede anvendelse af denne kraftfulde analyse bygger på et par…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).
Materials
| HEK293 cell line | |||
| DMEM/High glucose | Hyclone | SH30022.01 | High glucose 4.0 mM L-glutamine |
| Foetal Bovine Serum (FBS) | PuFei | 1101-100 | |
| Phosphate Buffered Saline (PBS) | Genom | GNM10010 | |
| Penicillin-Streptomycin | Genom | GNM15140 | |
| 0.25% Trypsin & 0.02% EDTA | Genom | GNM25200 | |
| Opti-MEM (1X) | GIBCO, by Life Technologies | 31985-062 | reduced serum media |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668027 | Reagent 1 |
| X-tremeGENE HP DNA Transfection Reagent | Roche | 6366236001 | Reagent 2 |
| DAPI Staining Solution | Beyotime | C1006 | |
| DiI | Beyotime | C1036 | |
| Antifade Mounting Medium | Beyotime | P0126 | |
| Paraformaldehyde | Sinopharm Chemical Reagent Co.(SCRC) | 80096618 | |
| 100 mm cell culture | CORNING | 430167 | |
| 6-well plate | ExCell Bio | CS016-0092 | |
| 12-well plate | ExCell Bio | CS016-0093 | |
| Cover Glass | NEST | 801007 | |
| Microscope Slides | Citoglas | 10127105P-G | |
| Thermo Scientific Forma CO2 Incubator | Thermo | 3111 | |
| TCS SP5II laser scanning confocal microscope | Leica | 5100001311 |
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