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. Hücre Hazırlanması <ol><li> Hücre kurtarma <ol><li> 37 ° C su banyosu için bir sıvı azot tankından dondurularak korunmuş insan embriyonik böbrek 293 (HEK293) hücreler aktarın ve 1 sürekli çalkalama - 2 dakika. </li><li> 10 cm&#39;lik bir hücre kültür tabağına dengelenmiş büyüme ortamı 10 mL (Dulbecco değiştirilmiş Eagle ortamı (DMEM),% 10 fetal sığır serumu (FBS) ve% 1 penisilin-streptomisin solüsyonu) eklenir. Yavaşça dengelenmiş bir büyüme ortamına çözülm?...

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Burada sunulan protokol HEK293 hücrelerinde hücre içi lokalizasyonu ve eksprese GPCR füzyon proteininin internalizasyonunu tespit etmek için etkin ve kolay yorumlanabilir bir yöntem sağlar. Teknik kolayca HEK293 ve BMN hücreleri 16 Bombyx mori kaynaklı corazonin reseptörünün hücresel yerini ve internalizasyon için birçok farklı gen ve hücre tipleri için uyarlanabilir. Bu güçlü testin başarılı uygulama birkaç anahtar faktöre dayanır....

Hücreler taşımak için yük trafik mekanizmalara sahip hücre dışı malzemeler-tür ligandlar, mikroorganizmalar, besin ve zar-ötesi proteinden-bir bilgi, enerji ve diğer amaçlar 1, 2 hücre olarak. Hücresel homeostazı, doku işlevinin ve genel hücrenin hayatta kalması için kritik öneme sahiptir. Floresan füzyon proteinlerinin hücre bazlı ekspresyon yolları 3 sinyal lokalizasyonu ve G-protein bağlı reseptörler (GPCR), transmembran reseptörleri, internalizasyonunu incelemek için etkili bir yoldur. Doğrudan floresans saptama teknikleri ile bir arada Aequorea victoria akvaryum balığındaki yeşil floresan protein (GFP) ile etiketlenmiş füzyon proteinlerinin ekspresyonu, geniş kabul görmüştür protein gibi araştırma 4, 5, 6. GFP tabanlı Detectiüzerinde sabitleme eserler 7 kaçınırken yaşayan hücrelerin gerçek zamanlı görüntüleme izin verme avantajına sahiptir. Çok yönlü klonlama vektörleri bir dizi, 9, çeşitli hücrelerde 8 GFP protein füzyonlarının ekspresyonunu kolaylaştırmak için inşa edilmiştir. pEGFP-N1 vektörü memeli hücrelerinde daha parlak floresans ve daha yüksek ifadesi için doğal tipte GFP arasından optimize edilmiş olan bir arttırılmış yeşil fluoresan protein (EGFP) kodlayan yaygın olarak kullanılan ve ticari olarak plazmid vektörüdür. EGFP flüoresan sinyal memeli hücrelerinde ifade gözlemlemek için, floresans mikroskopisi tercihen eş odaklı mikroskopi ile 488 nm&#39;de eksitasyon ve 507 nm&#39;de emisyon ile yapılmalıdır. Hücre içi lokalizasyonu ve reseptörlerin ligand aracılı içselleştirme İzleme GPCR&#39;lerin 10 sinyal iletimini ve araştırmak üzere yaygın bir tekniktir </s11,&gt; yukarı. Bir çok durumda, internalizasyonunu GPCR&#39;lerin duyarsızlaştırma (agonistlerin mevcudiyetinde rağmen teşvik cevabının hafifleme) 12, 13 neden olmaktadır. içsel reseptörleri lizozom dahil edilmiş ve bozulmuş ya da reseptörler ve deneylerde kullanılan hücre çizgilerinin yapısına bağlı olarak, hücre yüzeyine geri geri dönüştürülebilir edilebilir. Gonadotropin salgılatıcı hormon reseptörleri (GnRHRs) GPCR familyasına aittir ve hücre dışı bir amino-terminali, bir hücre içi -COOH terminal ile, tipik bir GPCR protein yapısına sahip ve yedi zar-ötesi (TM) 14 alanlarla kullanılabilir. (Sepiella japonica gelen GnRHR geni ile) yeniden birleştirici plazmit Sj GnRHR-EGFP transfeksiyonu ve (HEK293 hücrelerinde ifade edilir) EGFP-etiketli Sj GnRHR konfokal mikroskopi tespit Previou bildirilmiştirsinsi ve GFP floresan transmembran protein lokalizasyonu deneyleri 15 muhabir olarak kurulmuştur. Şimdi, S. japonica gonadotropin salgılatıcı hormon (GnRH Sj) ile aktive Sj GnRHR içselleştirilmesi, eş odaklı mikroskopi ile zaman ve doza bağlı şekillerde gösterilmiştir.

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			<td>High glucose 4.0 mM L-glutamine</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>
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			<td>Reagent 1</td>
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			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
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			<td>Reagent 2</td>
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			<td style="width:236px;">Beyotime</td>
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			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
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			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</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>
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			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<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>
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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>
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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>
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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.

Şekil 1, bir eş odaklı mikroskop sisteminin bir örneğini göstermektedir. Şekil 2, HEK293 hücrelerinde pEGFP-N1 ekspresyonunu göstermektedir. GFP sinyali sitoplazma ile çekirdek saptandı. Şekil 3, daha önce yayınlanmış sonuçlar 15 ile uyumlu HEK293 hücrelerinde S. japonica gelen GnRHR hücre içi lokalizasyonu göstermektedir. Zaman CLSM altında 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

Bu protokol, memeli hücrelerinde G-protein kenetli reseptör (GPCR) içselleştirmenin konfokal mikroskopi bağlanmasını anlatır. Temel hücre kültürü, transfeksiyon, ve konfokal mikroskopi prosedürü içeren füzyon ifade GPCR hücre içi lokalizasyonu ve içselleşmesini tespit etmek için etkin ve kolay yorumlanabilir bir yöntem sağlar.

Aynı odaklı mikroskopi ile ligand-aktif G protein-eşli reseptör özümseme tespiti

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.

Research

JoVE Journal

Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Konfokal Mikroskop Zebra balığı Embriyonik Beyin Görüntüleme Canlı

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biyoloji

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

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

Işıksal Çözülmüş İki foton Mikroskop kullanarak G Protein Coupled Reseptör Etkileşmeleri in vivo Niceleme

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

Görüntüleme G-protein Coupled Reseptör (GPCR) aracılı Sinyal Olaylar Kontrolü Kemotaksis Diktiyostelyum diskodeyum</em

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

G Protein-Coupled reseptörleri Agonist Aktivitesi miktarının

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

Genetik olarak kodlanmış Molecular Probes G protein-bağlanmış alıcılar Çalışması için

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

Bir floresan bazlı Kalsiyum Mobilizasyon Deneyi G proteini ile birleştirilmiş alıcıların karakterizasyonu

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

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

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

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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

TIRF Mikroskopi ile Tek olay ebatları da G protein-bağlanmış alıcılar üzerinden Klatrin dolayımlı endositoz görselleştirme

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

Colocalizational Analizi Reseptör Sonrası içselleştirme Ticareti Uyuşturucu kaynaklı Değişiklikleri İzleme

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