Name: detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy
Uploaded: 2017-04-09T14:00:00.000+00:00
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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％CO 2 2的潮湿气氛中- 4小时。 </li><li> 2个后 - 4小时，检查该细胞附着。替换另外24平衡的生长培养基和培养 - 48小时。 注意:因为它消除二甲亚砜（DMSO），并防止细胞损伤改变平衡生长介质是有用的。 </li></ol></li><li> &lt;强&gt;细胞通道<ol><li> 24个后 - 48小时，当细胞已经生长到接近汇合，弃去平衡生长培养基并用2ml磷酸盐缓冲盐水（PBS）洗涤细胞。轻轻摇动菜。 </li><li>弃去PBS和加入1毫升胰蛋白酶-EDTA（盐水，0.25％胰蛋白酶和0.02％EDTA）。以完全消化细胞，小心摇动培养皿中并吸脱胰蛋白酶-EDTA溶液。放于37℃的水夹套CO 2培养箱培养皿3分钟。 注意:在此期间，准备了三个新的10cm细胞培养皿，并添加10毫升平衡生长培养基到每个。 </li><li>使用光学显微镜，确定是否在细胞已经从培养皿的底部升起。加入2毫升平衡生长培养基的培养皿中。通过移液混合，并立即吸管0.6毫升细胞悬浮液的每一个的新菜肴。轻轻用移液管混合并允许细胞在37℃和5％CO 2孵育。 </li></ol></l我&gt; </ol> 2.细胞接种<ol><li> 24个后 - 48小时，当传代的细胞已经生长到接近汇合，弃去平衡生长培养基，并用2毫升的PBS洗涤细胞。轻轻摇动菜。 </li><li>弃去PBS和加入1毫升胰蛋白酶-EDTA。以完全消化细胞，小心摇动培养皿中并吸脱胰蛋白酶-EDTA。放于37℃的水夹套CO 2培养箱培养皿3分钟。 注意:在此期间，加入2毫升平衡生长培养基到每个孔中制备一个6孔板中。 </li><li>加入1毫升平衡生长培养基的菜，如果，在显微镜观察下，将细胞已经从培养皿底部升起。通过移液混合，并立即吸管0.1毫升细胞悬浮液（体积根据细胞生长密度调节），以所制备的6孔板（用2mL平衡生长培养基的细胞较少6孔板）的每个孔中。轻轻吹打混合，并允许细胞在37℃下温育和5％CO 2。 </li></ol> 3.细胞转染<ol><li> 转染方法1，用脂质体制剂 注:请参阅材料的表为转染试剂1。 <ol><li>次日，确保细胞应该是85 - 95％汇合。 </li><li>添加质粒DNA（500毫微克/微升）和100μL减少血清培养基中的3微克到无菌微量离心管中，并轻轻混合。孵育在室温下5分钟。 </li><li>制备9μL试剂1和在另一微量离心管中加入100μL低血清培养基中，并轻轻混合。孵育在室温下5分钟。 </li><li>结合用稀释的试剂1（总体积= 215微升）稀释的质粒混合物。轻轻混匀并孵育在室温下20分钟。 </li><li>吸出培养基断细胞培养板，并加入1.5毫升新鲜不含FBS的DMEM。 </li><li>添加DNA-TRANSF的215微升挠度试剂混合物到每个孔中的溶液，并通过旋动板轻轻混匀。 </li><li>孵育在37℃和5％CO 2的4 - 8小时。用新鲜的含10％FBS更换。 </li><li>孵育细胞在37℃和5％CO 2的另一个24 -运行的任何实验之前48小时。 </li></ol></li><li> 转染方法2，用脂质体制剂 注:请参阅材料的表为转染试剂2。 <ol><li>添加质粒DNA的2微克（500毫微克/微升），试剂2 4μL，和100μL减少血清培养基到无菌微量离心管中。轻轻混匀并孵育在室温下15分钟。 </li><li>将转染混合物加入到细胞中以逐滴方式。 </li><li>转染后，孵育细胞24 -在37℃下48小时和5％CO 2，在运行任何实验之前。 </li></ol></li></ol> 4.在盖玻片上的细胞培养<ol><li>回覆移动从培养6孔板的生长培养基中。 </li><li>洗用1mL PBS的细胞单层没有钙和镁。 </li><li>添加0.5毫升胰蛋白酶-EDTA。小心摇动培养皿中并吸弃胰蛋白酶-EDTA。孵育培养板在37℃下3分钟和5％CO 2。实际的培养时间与所使用的细胞系不同而不同。 注意:在此期间，准备一个新的12孔板，并添加直径15mm无菌盖玻片到每个孔中。涂有赖氨酸，层粘连蛋白或胶原盖玻片可以改善HEK293细胞容易脱离细胞附着。 </li><li>当细胞被分离，添加预热的1毫升完全生长培养基到每个孔中。通过移液混合，并立即将所需数量的细胞与盖玻片转移至12孔板。新鲜的完全的生长培养基添加到每个孔中以调节总体积至1mL </li><li>孵育细胞16 -在37℃下24小时和5％CO 2。 注:30后 -60分钟后，大部分细胞将已经粘附到无菌盖玻片上。 </li></ol> 5.共聚焦显微镜检测<ol><li> 细胞定位 <ol><li> 16个后 - 24小时，当细胞已经生长到接近汇合于在12孔板中的无菌盖玻片，添加膜探针的DiI，向每个孔中，得到的5μM的终浓度。使细胞温育在37℃下30分钟和5％CO 2。 </li><li> 30分钟后，取出平衡生长培养基并在冷PBS中洗涤细胞两次（2 - 8℃）。 </li><li>通过加入1毫升4％的多聚甲醛溶液（在PBS中）到每个孔中固定细胞。轻轻搅动15分钟。 注意:多聚甲醛是通过皮肤接触或吸入有中等毒性，并指定为可能的人类致癌物。化学品通风柜，排出平衡外壳或其它保护性措施应的多聚甲醛的称重和处理过程中被使用。&lt;/ LI&gt; <li>洗固定细胞两次在PBS中。添加200-300 DAPI（4&#39; ，6-二脒基-2-苯基吲哚）溶液（0.5微克/毫升）的μL到每个孔中，并孵育在室温下10分钟。 </li><li>用PBS洗细胞两次。小心地从板上取下盖玻片和灯芯掉过量的PBS。倒置到装载有安装平台（50％v / v甘油/水）一滴显微镜载玻片上。从载玻片移除过量的安装平台。 </li></ol></li><li> 细胞内 <ol><li> 16个后 - 24小时，当细胞已经生长到接近汇合于在12孔板中的无菌盖玻片，取出平衡生长培养基并加入新鲜的，温热的DMEM培养基（37℃）。孵育所述细胞在37℃下1个小时和5％CO 2。 </li><li>处理细胞用不同浓度（0，10 -12，10 -9，和10 -6 M）的配体的30分钟，并用10 -6 M的配体不同的时间（0对待它们，10，30和60分钟）。 </li><li>清洗细胞两次在冷PBS（2 - 8℃）。通过加入1毫升4％的多聚甲醛溶液（在PBS中）到每个孔中固定细胞。轻轻搅动15分钟。 </li><li>清洗细胞两次在PBS中。小心地从板上取下盖玻片和灯芯掉过量的PBS。倒置到装载有安装平台（50％v / v甘油/水）一滴显微镜载玻片上。从载玻片移除过量的安装平台。 </li></ol></li><li> 共聚焦显微镜 <ol><li>打开共焦显微镜的硬件，包括汞灯功率，PC显微镜，扫描仪和激光。登录到操作系统帐户，启动软件，检查配置和选择激光。按照说明书的顺序打开激光共聚焦显微镜系统的不同单元。 </li><li>放置制备玻片上的显微镜载物台上。在使用阶段控制器物镜样本位置。加上雪松油一滴被选择的油浸镜头时盖玻片。将盖玻片上面对镜头的幻灯片。 </li><li>找到感兴趣的细胞中的荧光显微镜下。 </li><li>切换到扫描模式。选择激光功率和发射荧光团的光谱范围。捕获由调谐激光强度和其它参数的高质量的共聚焦图像。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

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。

<table><tbody><tr><td>HEK293 cell line&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>DMEM/High glucose</td><td>Hyclone</td><td>SH30022.01</td><td>High glucose 4.0 mM L-glutamine</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>PuFei</td><td>1101-100</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Genom</td><td>GNM10010</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Genom</td><td>GNM15140</td><td></td></tr><tr><td>0.25% Trypsin &amp;amp; 0.02% EDTA</td><td>Genom</td><td>GNM25200</td><td></td></tr><tr><td>Opti-MEM (1x)</td><td>GIBCO, by Life Technologies</td><td>31985-062</td><td>reduced serum media</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen</td><td>11668027</td><td>Reagent 1</td></tr><tr><td>X-tremeGENE HP DNA Transfection Reagent</td><td>Roche</td><td>6366236001</td><td>Reagent 2</td></tr><tr><td>DAPI Staining Solution</td><td>Beyotime</td><td>C1006</td><td></td></tr><tr><td>DiI</td><td>Beyotime</td><td>C1036</td><td></td></tr><tr><td>Antifade Mounting Medium</td><td>Beyotime</td><td>P0126</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent Co.(SCRC)</td><td>80096618</td><td></td></tr><tr><td>100 mm cell culture</td><td>CORNING</td><td>430167</td><td></td></tr><tr><td>6-well plate&amp;nbsp;</td><td>ExCell Bio</td><td>CS016-0092</td><td></td></tr><tr><td>12-well plate&amp;nbsp;</td><td>ExCell Bio</td><td>CS016-0093</td><td></td></tr><tr><td>Cover Glass</td><td>NEST</td><td>801007</td><td></td></tr><tr><td>Microscope Slides</td><td>Citoglas</td><td>10127105P-G</td><td></td></tr><tr><td>Thermo Scientific Forma CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo</td><td>3111</td><td></td></tr><tr><td>TCS SP5II laser scanning confocal microscope</td><td>Leica&amp;nbsp;</td><td>5100001311</td><td></td></tr></tbody></table>

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 ...

detection-ligand-activated-g-protein-coupled-receptor-internalization

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.7K 次观看.  Zhejiang Ocean University. 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。 

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

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

科研

化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

神经科学

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

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 applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

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

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

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 ...

生物学

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 chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

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

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

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 ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

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

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

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 ...

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

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

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

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and 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 ...