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. خلية التحضير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> انتعاش خلية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نقل الجنينية الكلى 293 (HEK293) الخلايا البشرية cryopreserved من خزان النتروجين السائل ل37 ° C حمام الماء ويهز مستمر 1 - 2 دقيقة. </li><li style=";text-align:right...

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بروتوكول المعروضة هنا يوفر وسيلة فعالة وللتفسير بسهولة للكشف عن توطين التحت خلوية واستيعاب أعرب GPCR انصهار بروتين في الخلايا HEK293. هذه التقنية يمكن تكييفها بسهولة للعديد من جينات مختلفة وأنواع الخلايا، مثل لتوطين الخلوية واستيعاب مستقبلات corazonin من دودة القز في HE...

خلايا تمتلك البضائع الآلات الاتجار لنقل مواد مثل بروابط، والكائنات الدقيقة، والمغذيات، والبروتينات في الغشاء الخلية للحصول على معلومات، والطاقة، وغيرها من الأغراض 1، 2 خارج الخلية. ومن الأهمية بمكان للالتوازن الخلوي، وظيفة الأنسجة، وبقاء الخلية بشكل عام. التعبير القائم على خلية من البروتينات الانصهار فلوري هو وسيلة قوية لدراسة توطين واستيعاب مستقبلات الغشاء، مثل G مستقبلات البروتين يقترن (GPCR)، في مسارات الإشارات 3. وقد اكتسب التعبير عن البروتينات الانصهار ذات الكلمات الدلالية مع الأخضر بروتين فلوري (GFP) من فيكتوريا قنديل البحر Aequorea، جنبا إلى جنب مع تقنيات الكشف عن مضان المباشر، قبولا واسعا في استهداف بروتين البحوث 4، 5، 6. detecti القائم GFPعلى ديها ميزة تسمح للتصوير في الوقت الحقيقي من الخلايا الحية مع تجنب القطع الأثرية تثبيت 7. وقد تم بناؤها سلسلة من ناقلات الاستنساخ متعددة لتسهيل التعبير عن اندماج البروتين GFP في مختلف خلايا 8 و 9. متجه pEGFP-N1 هو متجه البلازميد تستخدم على نطاق واسع وتسويقها ترميز لتعزيز البروتين الفلوري الأخضر (EGFP)، والتي تم الأمثل من النوع البري GFP لأكثر إشراقا مضان والتعبير العالي في خلايا الثدييات. لمراقبة أعربت إشارة الفلورسنت من EGFP في خلايا الثدييات، ينبغي إجراء الفحص المجهري مضان مع الإثارة في 488 نانومتر، وانبعاث في 507 نانومتر، ويفضل مع المجهر متحد البؤر. رصد توطين التحت خلوية واستيعاب بوساطة يجند مستقبلات هي تقنية مشتركة للتحقيق في نقل الإشارة وظيفة GPCRs 10 </sتصل&gt; 11. في معظم الحالات، واستيعاب يؤدي إلى الحساسية من GPCRs (تخفيف من الاستجابة التحفيز على الرغم من وجود منبهات) 12، 13. ويمكن إدراج المستقبلات المنضوية في يحلول والمتدهورة، أو أنها يمكن إعادة تدويرها مرة أخرى إلى سطح الخلية، وهذا يتوقف على طبيعة المستقبلات وخطوط الخلايا المستخدمة في التجارب. مستقبلات هرمون موجهة الغدد التناسلية الافراج عن (GnRHRs) تنتمي إلى عائلة GPCR ولديها بنية البروتين GPCR نموذجي، مع محطة الأمينية خارج الخلية، محطة -COOH داخل الخلايا، وسبعة الغشاء (TM) 14 المجالات. وأفاد ترنسفكأيشن البلازميد المؤتلف صج GnRHR-EGFP (مع الجين GnRHR من Sepiella تفرضه اليابان) وكشف الفحص المجهري متحد البؤر من الموسومة EGFP صج GnRHR (معبرا في الخلايا HEK293) بريفيوخبيث، وتأسست مضان GFP كمراسلة للبروتين الغشاء فحوصات توطين 15. الآن، واستيعاب صج GnRHR، تفعيلها من خلال S. الجابونيكا بين الافراج عن هرمون موجهة الغدد التناسلية (SJ نره])، وقد تجلى في الأدب زمنية وتعتمد على الجرعة عن طريق الفحص المجهري متحد البؤر.

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			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
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			<td style="width:119px;">1101-100</td>
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			<td>Reagent 2</td>
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			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
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			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
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			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
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			<td style="width:119px;">CS016-0093</td>
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			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
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			<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>
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			<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.

ويبين الشكل 1 مثالا لنظام المجهر متحد البؤر. ويبين الشكل 2 التعبير عن pEGFP-N1 في الخلايا HEK293. تم الكشف عن إشارة GFP في السيتوبلازم والنواة. ويبين الشكل 3 توطين التحت خلوية من GnRHR من S. تفرضه اليابان في الخلايا HEK293، بما يتفق مع...

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

يصف هذا البروتوكول كشف الفحص المجهري متحد البؤر من مستقبلات البروتين يقترن-G (GPCR) تدخيل في خلايا الثدييات. ويشمل الثقافة الأساسية الخلية، ترنسفكأيشن، وإجراء الفحص المجهري متحد البؤر، ويوفر وسيلة فعالة وللتفسير بسهولة للكشف عن توطين التحت خلوية واستيعاب GPCR-أعرب الانصهار.

الكشف عن تنشيط يجند G-إلى جانب البروتين مستقبلات التطبع التي كتبها متحد البؤر المجهري

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

يعيش تصوير الدماغ الجنينية اسماك الزرد بواسطة الميكروسكوب متحد البؤر

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 ...

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

الكمي في الجسم الحي من التفاعلات بروتين G مستقبلات اقترن به الطيفي ثنائي الفوتون الميكروسكوب

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

التصوير G - مستقبلات البروتين يقترن (GPCR) بوساطة الأحداث يشير إلى أن التحكم في الانجذاب الكيميائي Dictyostelium Discoideum</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

قياس نشاط ناهض في مستقبلات البروتين يقترن G

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

المسابر الجزيئية المشفرة وراثيا لدراسة G مستقبلات البروتين يقترن

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

توصيف G مستقبلات البروتين يقترن بها الكالسيوم تعبئة الفحص القائم على الإسفار

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

في الجسم الحي تتبع نسيلي من الخلايا الجذعية المكونة للدم والسلف تميزت خمسة البروتينات الفلورية باستخدام الميكروسكوب متحد البؤر وMultiphoton

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

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

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

تصور الإلتقام بالكلاذرين بوساطة من G مستقبلات البروتين يقترن قرار واحد في الحدث عبر الميكروسكوب TIRF

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

تتبع التغييرات الناجمة عن المخدرات في مستقبلات بعد استيعاب الاتجار عن طريق تحليل Colocalizational

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