Name: detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy
Uploaded: 2017-04-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

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).

1. Cellefremstilling <ol><li> Cell utvinning <ol><li> Overfør kryokonserverte humane embryoniske nyre 293 (HEK293) celler fra en flytende nitrogentanken til et 37 ° C vannbad, og det hele rystes kontinuerlig i 1 - 2 min. </li><li> Tilsett 10 ml ekvilibrert vekstmedium (Dulbeccos modifiserte Eagles medium (DMEM), 10% føtalt bovint serum (FBS), og 1% penicillin-streptomycin-løsning) til en 10-cm cellekulturskål. Tilsett den opptinte celler til den ekvilibrerte vekstmediet. </li><li> Cellene inkuber...

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Protokollen som presenteres her tilveiebringer en effektiv og lett tolkbar metode for å påvise subcellulære lokalisering og internalisering av uttrykt GPCR fusjonsprotein i HEK293-celler. Teknikken kan lett tilpasses til mange forskjellige gener og celletyper, slik som for cellulær lokalisering og internalisering av den corazonin reseptoren fra Bombyx mori i HEK293 og BMN celler 16. Vellykket bruk av dette kraftige analysen bygger på noen viktige faktore...

Celler som har last trafikkmaskiner for transport av ekstracellulære materialer, for eksempel ligander, mikroorganismer, næringsstoffer og transmembranproteiner-inn i cellen for å få informasjon, energi, og andre formål 1, 2. Det er viktig for cellulær homeostase, vev funksjon, og total celleoverlevelse. Den cellebaserte ekspresjon av fluorescerende fusjonsproteiner er en effektiv måte å studere lokalisering og internalisering av transmembranreseptorer, slik som G-protein-koblede reseptorer (GPCR), i signaliseringsveier 3. Ekspresjon av fusjonsproteinene kodet med grønt fluorescerende protein (GFP) fra maneter Aequorea Victoria, kombinert med direkte fluorescens-deteksjonsteknikker, har fått bred aksept i protein-målsøkende forskning 4, 5, 6. GFP-baserte detectividere har den fordel at den tillater sanntids avbildning av levende celler mens man unngår fikseringsartifakter 7. En rekke allsidige kloningsvektorer er konstruert for å lette ekspresjonen av proteinfusjoner til GFP i forskjellige celler 8, 9. Den pEGFP-N1-vektoren er en utbredt og kommersialisert plasmid-vektor som koder for en forbedret grønt fluorescerende protein (EGFP), som har blitt optimalisert fra villtype-GFP for lysere fluorescens og høyere ekspresjon i pattedyrceller. For å observere det fluorescente signalet av EGFP uttrykt i pattedyrceller, bør den fluorescens mikroskopi utføres med eksitasjon ved 488 nm og emisjon ved 507 nm, fortrinnsvis med konfokal mikroskopi. Overvåking av subcellulære lokalisering og ligand-mediert internalisering av reseptorene er en vanlig teknikk for å undersøke signaloverføring og funksjon av GPCR 10 </sopp&gt;, 11. I de fleste tilfeller fører til internalisering desensibilisering av GPCR (dempningen av stimulus respons på tross av nærvær av agonister) 12, 13. De internalisert reseptorene kan bli innlemmet i lysosomet og brytes ned, eller de kan resirkuleres tilbake til celleoverflaten, avhengig av naturen av reseptorene og cellelinjene anvendt i eksperimentene. De gonadotropinfrigjørende hormon-reseptorer (GnRHRs) tilhører den GPCR familien og har en typisk GPCR proteinstruktur, med et ekstracellulært aminoterminal, en intracellulær -COOH terminal, og syv transmembran (TM) domener 14. Den transfeksjon av rekombinant plasmid Sj GnRHR-EGFP (med GnRHR genet fra Sepiella japonica) og den konfokal mikroskopi påvisning av EGFP-merket Sj GnRHR (uttrykt i HEK293 celler) ble rapportert previouslu, og GFP fluorescens ble etablert som en reporter for transmembranprotein lokaliserings analyser 15. Nå, internalisering av Sj GnRHR, aktivert av S. japonica gonadotropin-frigjørende hormon (GnRH Sj), ble demonstrert i tids- og doseavhengig oppførsel ved hjelp av konfokal mikroskopi.

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			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
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			<td style="width:203px;"></td>
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			<td height="21" style="height:21px;width:300px;">DMEM/High glucose</td>
			<td style="width:236px;">Hyclone</td>
			<td style="width:119px;">SH30022.01</td>
			<td>High glucose 4.0 mM L-glutamine</td>
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			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
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			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
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			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
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			<td height="21" style="height:21px;width:300px;">Opti-MEM (1X)</td>
			<td style="width:236px;">GIBCO, by Life Technologies</td>
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			<td>reduced serum media</td>
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			<td height="24" style="height:25px;width:300px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td>Reagent 1</td>
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		<tr height="24">
			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
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			<td height="22" style="height:22px;width:300px;">DAPI Staining Solution</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1006</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
			<td></td>
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			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
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			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
			<td style="width:236px;">CORNING</td>
			<td style="width:119px;">430167</td>
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			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
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			<td style="width:119px;">CS016-0092</td>
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			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
			<td></td>
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			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
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			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
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			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
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			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">5100001311</td>
			<td></td>
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detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

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.

Figur 1 viser et eksempel på et system konfokalt mikroskop. Figur 2 viser ekspresjon av pEGFP-N1 i HEK293-celler. GFP signal ble detektert i cytoplasma og cellekjernen. Figur 3 viser subcellulære lokalisering av GnRHR fra S. japonica i HEK293 celler, i tråd med våre tidligere publiserte resultater 15. Fusjonsproteinet med EGFP-koden i den C-terminale ende av Sj GnRHR (Sj GnRHR-EGFP) ...

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Denne protokollen beskriver konfokal mikroskopi påvisning av G-protein-koblet reseptor (GPCR) internalisering i pattedyrceller. Den inneholder den grunnleggende cellekultur, transfeksjon, og konfokal mikroskopi og fremgangsmåte tilveiebringer en effektiv og lett tolkbar metode for å påvise subcellulære lokalisering og internalisering av fusjons uttrykt GPCR.

Påvisning av ligand-aktiverte G-protein-koblet reseptor Internalisering ved hjelp av konfokal mikroskopi

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

The overall goal of this experiment is to provide an efficient and easily-interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion proteins in mammalian cells. This method can help answer the key questions in the subcellular protein localization field, such as receptor internalization and recycling. The main advantage of this technique is that it can be used easily and efficiently.So this method can provide insight in monitoring and locating GPCRs. And it can also apply to other systems, such as cytoplasmic and nuclear protein. After recovering and passaging HEK293 cells according to the text protocol, when the cells have grown to near confluence, discard the equilibrated growth medium, add two milliliters of PBS, and swirl the dish to wash the cells.Discard the PBS, and add one milliliter of trypsin EDTA. To completely digest the cells, carefully swirl the dish and aspirate off the solution. Then, put the dish in this 37 degrees Celsius water-jacketed C02 incubator for three minutes.While the cells are incubating, prepare a six well plate by adding two milliliters of equilibrated growth medium to each well. Then, when the cells have lifted from the bottom of the dish, add one milliliter of equilibrated growth medium to the dish. Mix by pipetting and immediately transfer 0.1 milliliters of cell suspension into each well of the prepared six well plate.Gently pipette the suspension in the wells, and incubate the cells at 37 degrees Celsius and 5%CO2. The following day, ensure that the cells are 85-95%confluent. Then, to two sterile microcentrifuge tubes, add three micrograms of plasmid DNA and 100 microliters of reduced serum medium to one tube.Meanwhile, add nine microliters of reagent one and 100 microliters of reduced serum medium to another tube. Incubate the tubes at room temperature for five minutes. Combine the diluted plasmid mixture with diluted reagent one.Then, gently mix the solution, and incubate it at room temperature for 20 minutes. Aspirate the medium of the cells and add 1.5 milliliters of fresh DMEM without FBS. Then, add dropwise 215 microliters of DNA transfection reagent mixture to each well, and gently mix by swirling the plate.Incubate the plate at 37 degrees Celsius and 5%CO2 for four to eight hours. Then, replace the medium with fresh DMEM with 10%FBS. Incubate the cells for another 24 to 48 hours before running any experiments.Transfection efficiency may vary greatly in different cell lines. Therefore, the optimization of the sit and stab in deposition may be necessary depending on the particular cell type used. Remove the growth medium from the six well culture plate.Then, use one milliliter of PBS to wash the cell monolayer. Add 0.5 milliliters of trypsin EDTA, swirl the dish, and aspirate off the solution. Then, incubate the culture plate at 37 degrees Celsius for three minutes.During the incubation, insert 15 millimeter diameter sterile coverslips into each well of a 12 well plate. Resuspend the cells in a small volume of fresh complete growth medium, and transfer the required number of cells to the 12 well plate with coverslips. Then, incubate the cells for 16 to 24 hours.After 16 to 24 hours of incubation, when the cells have grown to near confluence, add five micromolar of the membrane probe dill to each well, and place the cells back in the incubator for 30 minutes. Remove the equilibrated growth medium and use cold PBS to wash the cells twice. Then, add one milliliter of 4%PFA to each well, and agitate the cells for 15 minutes to fix the cells.With PBS, wash the fixed cells twice, then add 200 to 300 microliters of DAPI to each well, and incubate the plate for 10 minutes at room temperature. After washing the cells with PBS twice, carefully remove the coverslips from the plate, and wick off the excess buffer. Invert the coverslips onto microscope slides loaded with one drop of mounting medium.Then, remove the excess mounting medium from the slides. Using cells that have grown to near confluence, remove the equilibrated growth medium and add fresh warmed DMEM. Incubate the cells for one hour.Treat the cells with different concentrations of ligand for 30 minutes. And treat the cells with 10 to the negative six molar ligand for the different times shown here. Use cold PBS to wash the cells twice, then add one milliliter of 4%PFA in PBS to each well, before gently agitating the cells for 15 minutes.After washing the cells twice in PBS, carefully remove the coverslips from the wells of the plate, and mount them as just demonstrated earlier in this video. To carry out confocal microscopy, turn on the hardware of the confocal microscope, including the mercury lamp power, PC microscope, scanner, and laser. Launch the software, then check the configuration and select the laser.Turn on the different units of confocal microscopy system according to the instructions. Place a prepared slide on the microscope stage, and use the stage controller to position the sample over the objective lens. For an oil immersion lens, add one drop of cedar oil on to the coverslip, and orient the slide with a coverslip facing the lens.Under fluorescence, find the cells of interest, then switch to scan mode, and choose the laser power and spectral range of the emission fluorophore. Capture high-quality images by tuning the laser intensity and other parameters. This figure illustrates the expression of pEGFP-N1 in HEK293 cells.The GFP signal was detected in the cytoplasm and the nucleus. The subcellular localization in HEK293 cells of gonadotropin-releasing hormone receptors from S-Japonica, or SjGnRHR, is shown here. EGFP receptors are in green, and Dil staining, which highlights the cell membrane, is in red.The receptors are seen localizing to the plasma membrane. To visualize the internalization of SjGnRHRs, cells were treated with gonadotropin-releasing hormone at different concentrations. As seen here, the internalization of the receptors proceeded in a dose-dependent manner, and the receptor was completely internalized from the cell's surface with the 10 to the minus six molar ligand.When treated with 10 to the minus six molar ligand for different time periods, internalized SjGnRHRs were detectable 10 minutes after agonist simulation, and extreme internalization occurred by 60 minutes. As demonstrated in HEK293 cells, in both experiments, the fluorescent SjGnRHR EGFP fusion protein primarily localized to the plasma membrane, and was dramatically and rapidly internalized into the cells. While attempting this procedure, it's important to remember to keep the cells under the right conditions without bacteria, fungi, and mycoplasma contamination.Following this procedure, and the methods like cell checking and immobilization can be performed in order to answer additional questions like the second in pass V of the protein receptors. After each development, the technology paved the way for researchers in the field of the cell biology and biochemistry to explore the localization and the internalization of transmembrane receptors in various cell lines.

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In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

Kvantifisering agonistaktivitet på G protein-koblede reseptorer

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Genetisk kodet Molekylær Sonder til Study G protein-koblede reseptorer

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Karakterisering av G-protein-koblede reseptorer ved en fluorescens-basert Kalsium Mobilisering analysen

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Klonal Sporing av Hematopoetiske Stem og stamceller Preget av fem Fluorescent Proteiner bruker Confocal og multiphoton Mikros

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Sporing av Hematopoietiske Stem og stamceller i Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Visualisere clathrin endocytose av G-protein-koblede reseptorer på enkelthendelse oppløsning via TIRF Mikros

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

Sporing Drug-induced Endringer i Receptor Post-intern Trafficking etter Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...