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.细胞的制备<ol><li> 细胞恢复 <ol><li>转移冷冻保存的人胚胎肾293（HEK293）细胞从液氮罐到37℃水浴中，并用于1连续摇 - 2分钟。 </li><li>加入10 mL的平衡的生长培养基（Dulbecco改良的Eagle培养基（DMEM），10％胎牛血清（FBS）和1％青霉素 - 链霉素溶液），以10厘米的细胞培养皿中。轻轻将解冻的细胞添加到平衡生长培养基。 </li><li>孵育细胞在37℃的水夹套CO 2培养箱用含95％空气和5...

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这里介绍的协议提供了一种有效和容易解释的方法来检测在HEK293细胞中的亚细胞定位和表达GPCR融合蛋白的内在化。该技术可以容易地适用于许多不同基因和细胞类型，诸如用于从家蚕在HEK293和家蚕细胞16 corazonin受体的细胞定位和内化。 这个功能强大的试验的成功应用依赖于几个关键因素。最重要的是细胞的身体健康，这是转染效率和数据质量至关重?...

细胞具有贩卖货物机械运送细胞外材料-如配体，微生物，营养素，和跨膜蛋白，成信息，能源和其他目的1，2中的单元格。它是细胞内环境稳定，组织的功能和整体细胞存活的关键。荧光融合蛋白的基于细胞的表达是一个功能强大的方法来研究的定位和跨膜受体，如G蛋白偶联受体（GPCR），内化在信号通路3。 从水母Aequorea victoria的标记有绿色荧光蛋白（GFP）的融合蛋白的表达，与直接荧光检测技术相结合，在已获得广泛的接受蛋白靶向研究4，5，6。基于GFP-detecti上具有允许活细胞，同时避免固定的工件7的实时成像的优点。一系列通用的克隆载体的已被构造成便于在各种细胞中8蛋白质融合到GFP的表达，9。的质粒pEGFP-N1载体是编码增强型绿色荧光蛋白（EGFP），它已被从野生型GFP优化用于在哺乳动物细胞更亮的荧光和更高的表达一种广泛使用的并商业化的质粒载体。观察EGFP的荧光信号在哺乳动物细胞中表达时，荧光显微术，应在488nm处激发和在507 nm处的发射，最好用共聚焦显微镜下进行。 监测的亚细胞定位和受体配体-介导的内是一种常用的技术来研究的GPCR 10的信号转导和功能</s向上&gt;，11。在大多数情况下，内化导致的GPCR脱敏（尽管激动剂的存在下的刺激反应的衰减）12，13。内化受体可以结合到溶酶体和降解，或它们可以被再循环回到细胞表面，这取决于受体和在实验中使用的细胞系的性质。 促性腺激素释放激素受体（GnRHRs）属于GPCR家族，并具有典型的GPCR蛋白结构，具有细胞外氨基末端，一个细胞内-COOH终端，和七个跨膜（TM）结构域14。重组质粒Sj的 GnRH受体-EGFP的转染（从曼氏粳稻的促性腺激素释放激素受体基因）和EGFP标记的GnRH受体Sj的 （在HEK293细胞中表达）的共焦显微镜检测报道上10sly和GFP荧光确立为跨膜蛋白定位试验15个记者。现在，SJ GnRH受体的内在化，通过S.粳稻促性腺激素释放激素（GnRH的Sj的 ）激活，表现在通过共聚焦显微镜时间和剂量依赖性。

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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 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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM10010</td>
			<td></td>
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			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM15140</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
			<td style="width:236px;">Genom</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>
			<td style="width:119px;">31985-062</td>
			<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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			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
		</tr>
		<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>
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		<tr height="42">
			<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>
			<td></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>
			<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>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
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		<tr height="21">
			<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.

图1显示了共焦显微镜系统的一个例子。 图2呈现的pEGFP-N1的在HEK293细胞中的表达。在细胞质和细胞核中检测到GFP信号。 图3显示了促性腺激素释放激素受体的从国槐在HEK293细胞中的亚细胞定位，与我们先前公布的结果一致15。该融合蛋白与GnRH受体Sj的 （Sj的 GnRH受体-EGFP）的C末端的EGFP标签在这个实...

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

这个协议描述了在哺乳动物细胞中的G蛋白偶联受体（GPCR）的内化共聚焦显微镜检测。它包括基本细胞培养，转染，和共聚焦显微镜的程序和提供了一个高效和容易解释的方法来检测融合表达GPCR的亚细胞定位和内化。

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

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

现场共聚焦显微镜对斑马鱼的胚胎脑成像

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 ...

科研

生物学

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

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

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

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</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

量化在G蛋白偶联受体激动剂活性

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

基因编码的分子探针来研究G蛋白偶联受体

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

G蛋白偶联受体的基于荧光的钙动员含量表征

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

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

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

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

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

追踪药物诱导Colocalizational变化分析受体后内部贩运

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