这项工作是由国立卫生研究院从NIAID的，壁间基金的支持。

1。 粘菌的趋化主管细胞的制备<ol><li>要生成四discoideum细胞趋化的趋化营，收获细胞增长，从一个惊天文化D3 - T的富媒体在22 ° C。 </li><li>非营养发育缓冲区清洗细胞两次（DB缓冲区含5毫米的Na 2 HPO 4，5毫米的KH 2 PO 4，MgCl 2的2毫米，0.1毫米氯化钙 2） 。 </li><li>重新暂停在DB缓冲区细胞密度在2 × 10 7细胞/ ml。 </li><li>摇在100转速在22 ° C 10毫升细胞一小时，在250毫升容量瓶中。 </li><li> 10毫升的细胞超过6小时，每6分钟提供100μL7.5μM的cAMP的股票达到了75纳米营的终浓度，cAMP的脉冲治疗指定的进程。经过5-6小时内cAMP脉冲治疗，D. discoideum细胞成为趋朝内cAMP梯度主管。 </li><li> 200克5分钟离心收集细胞，然后与数据库缓冲悬浮细胞含有咖啡因2.5毫米，摇匀，在200转，在22 ° C加热20分钟basolate细胞一个细胞趋化的形势。 </li></ol> 2。在可见的和可操作的趋化因子梯度成像chemotaxing细胞<ol><li>回填一个微管与新鲜配制的30μL1微米的营地和Alexa 5940.1μg/μl在DB缓冲区的解决方案。 </li><li>的Femtotip连接到一个微管人和连接管，压力供应装置的Eppendorf FemtoJet系统。 </li><li>将微量大会（Eppendorf公司TransferMan NK2）机动显微提供一个稳定的压力，以建立一个稳定的渐变到显微。 </li><li>一个6毫升数据库缓冲区填充超过40X石油镜头共聚焦显微镜以及LabTek室安装和使用的视野明亮场光学中心Femtotip。 </li><li>打开的供给压力，并设置为70百帕补偿的压力（PC），建立一个营/ Alexa的594混合的梯度。 </li><li>可视化监测所需的营地和Alexa 594与543 nm激光线激发荧光使用浓度的混合物中cAMP梯度。 </li><li>使用自动定位的显微操纵器的功能，把微量到所需的位置和设置位置1，位置2，位置3操纵细胞暴露梯度。 </li></ol> 3。不动无极细胞系统方便营梯度传感信号成像涉及事件<ol><li>咖啡因治疗后，取出在500克等分细胞和离心3分钟。 </li><li>取出缓冲和稀释细胞以5 × 10 5细胞/毫升新鲜的DB缓冲区含2.5毫米咖啡因。 </li><li>用1 ml的细胞悬液单以及商会或0.4毫升，每孔以及四室。 </li><li>让细胞坚持10分钟，仔细移液器关闭缓冲区，删除独立的细胞，并取代同体积。 </li><li>在显微镜下找到所需的细胞，并开​​始成像。 </li><li>监察动态信号在细胞暴露在一个稳定的梯度组件设计一个实验，把5.0微米（终浓度）Latrunculin乙前10分钟的实验细胞。 </li></ol> 4。同时监测异三聚体G蛋白的激活和PIP 3生产<ol><li> cAMP的脉冲培养细胞共同表达GαCFP和YFPGβ（G细胞）和细胞表达PIP 3指标的PH - GFP（PH值细胞）7。 </li><li>在1:1的比例和板他们在一孔或4孔商会混合这两种类型的细胞。 </li><li>创建和保存排放指纹参考曲线CFP，YFP和GFP与一个10纳米的宽度从464到624纳米的光谱范围内使用LAMBDA栈收购模式。 </li><li>同时图像G蛋白激活G细胞和PIP 3 PH值从464至544纳米与10纳米的增量的光谱范围内使用相同的Lambda堆栈采集模式的细胞生产。 </li><li>应用线性分解蔡司510META软件功能，使用保存CFPand YFP和排放指纹数学计算每个荧光在lambda协议栈的贡献，分离成单独的渠道在CFP和YFP的强度在G </li><li>同样的策略，适用于线性分解的功能，使用数学计算绿色荧光强度在PH细胞保存的GFP和背景排放指纹。 </li></ol> 5。代表性的成果： <ol><li> D 的一个很好的模型系统G蛋白偶联受体介导的趋化作用。discoideum社会变形虫，D. discoideum展品在生命周期的一个显着的趋化。由于它的遗传和生化优势，D. D提供了一个功能强大的System研究的趋化作用。 </li><li> 细胞趋化下一个可见的和可操作chemoattract的领域 ，在这里，我们首先展示一个简单的的方法获得cAMP浓度和稀释的2微米营的10微克/毫升混合系列荧光染料Alexa的594强度之间的线性关系Alexa的594（图2A）。下一步，我们提供了一个简单的方法来可视化的梯度，此外，为了建立一个渐变的cAMP浓度的定量测量的Alexa 594（图图2b）的强度。 </li><li> 细胞运动与细胞极化和定向感应耦合。肌动蛋白聚合抑制剂消除既存的形态学极性，也可以防止细胞的运动，同时保持细胞的定向感应能力（图3A） 。可见的和可操作的 cAMP刺激就业保障的投入，例如统一适用的刺激或渐变。此方法允许梯度传感机械营诱导的关键信号元件再分配的定量分析。这些信号组件测量的时空动态，让我们了解如何信令网实现适应统一的刺激而产生极化生化反应梯度（图3B）。 </li><li> 全身性的化学传感信号后，接触到一个稳定的梯度网络的动力学测量，这是关键的衡量动力学/动力学定向感应信号组件了解每个组件如何有助于建立细胞内极化时，细胞的经验第一梯度。活细胞成像中的应用与一个高节奏的空间分辨率，我们首先显示双相PIP3生产的细胞暴露在一个稳定的营梯度（图4A - C），。采用活细胞成像，我们有系统地测量的动态定向感应cAMP刺激PIP3的生产（图4D，E），具体的信令网。 </li><li> 同步监测异三聚体G蛋白的激活和画中画3条生产后，一律适用cAMP刺激。福斯特共振能量转移（FRET的缩写）提供了一种有效的方法，监察cAMP刺激后异三聚体G蛋白的激活（分解） 。在这里，我们描述了一个异三聚体G蛋白的激活和PIP 3生产的同时测量，方便易采取系统监测FRET变化和膜画中画易位 ，分别为3探头，PH值在G和PH细胞的GFP（图5 ）。一个统一适用的cAMP刺激触发一个持久的G蛋白的激活，而这将触发一个短暂的PIP 3生产。 </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="图1"/> 图1：D 的一个很好的模型系统discoideum为GPCR介导的趋化。 B. CAMP梯度诱导D.迅速趋化A.计划显示了一个简短的定向感应信号转导通路。 discoideum细胞。细胞表达画中画3探头，PH - GFP（绿色）。梯度（红色）594 Alexa的可视化。比例尺为50μm。 <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="图2"/> 图2：下一个可见的和可操作chemoattract的领域的细胞趋化。 答 ：图表显示cAMP浓度和强度的荧光染料Alexa的594系列稀释的2微米与10μg/ mL的Alexa的594混合的营地之间的线性关系。营梯度浓度的定量测量的线性关系cAMP浓度和强度的荧光染料Alexa的594 A中<img src="/files/ftp_upload/3128/3128fig3.jpg" alt="图3"/> 图3：细胞运动与细胞极化和定向感应耦合。 A.图像显示，动弹不得细胞治疗肌动蛋白聚合抑制剂Latrunculin乙保持定向检测的能力。细胞表达画中画3探头，PH - GFP（绿色）。渐变（红）594 Alexa的可视化。B.可操作的cAMP的刺激和不动的电池系统允许定向检测，以解决关键问题。比例尺=10μm的。 <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="图4"/> 图4：后，接触到一个稳定的梯度化学传感信号网络系统动力学测量 。 A.蒙太奇显示双相PIP 3条生产（绿色）的细胞接触到一个稳定的营梯度（红色）。图像显示在C。C画中画3条生产动力学测量地区的利益（投资回报） 。动力学直径f画中画3生产的细胞在接触到一个稳定的梯度。D.计划显示了从cAMP刺激画中画3条生产定向检测信令网。他们在接触到一个稳定的梯度动力学是在 E在相同颜色的实线。 <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="图5"/> 图5：同时监控多的G蛋白偶联受体信号网络事件。 A.计划显示监测FRET分别PIP3的探头，PH - GFP的G和PH细胞的变化和膜易位异三聚体G蛋白的激活和 PIP 3生产的同时测量B. G和PH细胞的彩虹图像蒙太奇显示。一个统一适用的cAMP刺激触发一个持久的G蛋白激活，在细胞外围，而这将触发一个短暂的PIP3的生产。时间点前（0）和刺激后4.9s，10.2s和20.4s。C. G蛋白激活后一个统一适用的cAMP刺激生产和PIP3的动力学。

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没有利益冲突的声明。

深远的趋化细胞的主管阶段进程对于野生型D. discoideum细胞，大约需要5〜6小时脉冲在室温下的发展，诱导他们进入一个良好的趋化主管的阶段，在此期间，细胞中显示良好的极化的细胞形态和细胞快速迁移（图1）。几个因素，如cAMP的浓度为脉冲，温度和不同的遗传背景，可能会影响深远的趋化主管阶段的过程。一个营地梯度引导细胞走向cAMP的来源，细胞迁移的趋化指定...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> D3 - T的生长介质</td><td> KD医疗</td><td></td><td></td></tr><tr><td>咖啡因</td><td>西格玛</td><td></td><td></td></tr><tr><td> Latrunculin乙</td><td>分子探针</td><td></td><td></td></tr><tr><td> Alexa的594 </td><td>分子探针</td><td></td><td></td></tr><tr><td>腺苷</td><td>西格玛</td><td></td><td></td></tr><tr><td> ChronTrol XT的可编程定时器</td><td> ChronTrol公司</td><td></td><td></td></tr><tr><td> Miniplus 3蠕动泵</td><td> Gilbson </td><td></td><td></td></tr><tr><td>平台旋转摇床</td><td></td><td></td><td></td></tr><tr><td> FemtoJet microcapillary供给压力</td><td> Eppendorf公司</td><td></td><td></td></tr><tr><td>单和四以及实验室康二玻璃罩商会</td><td>国际Nalge NUNC </td><td></td><td></td></tr><tr><td> LSM 510 META或有同等作用的荧光显微镜</td><td>蔡司</td><td></td><td>一个40X 1.3 NA或60X 1.4无油DIC的计划NEOFLUAR物镜</td></tr><tr><td>奥林巴斯X81或同等学历</td><td>奥林巴斯</td><td></td><td>需要一个100X 1.47 NA TIRF物镜</td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

许多真核细胞中可以检测到在其环境中的化学信号的梯度迁移相应的 1 。这种引导细胞迁移被称为趋化作用，这是必不可少的各种细胞进行他们的功能，如免疫细胞和神经细胞2，3的图案贩运。一个大家族的G蛋白偶联受体（GPCRs的）检测变量，作为趋化因子的小分子肽， 直接在 体内 4细胞的迁移。趋化研究的最终目的是要了解一个GPCR的机械感官的趋化因子梯度和控制信号的事件，以趋化。为此，我们使用成像技术，实时监测，时空趋化因子，细胞运动的趋化因子梯度浓度，G蛋白偶联受体介导的异三聚体G蛋白的激活，细胞内的信号在 5-8真核细胞趋化涉及事件。简单的真核有机体，粘菌，显示chemotaxic行为类似白细胞和 D discoideum是一个重要的模型系统，为研究真核细胞趋化。由于自由生活的阿米巴，D discoideum细胞分裂丰富培养基。饥饿时，细胞进入一个发展的计划中，他们通过cAMP介导的趋化聚集，形成multicullular结构。趋化阵营中涉及的许多组件已经确定在 D discoideum。阵营的一个G蛋白偶联受体的结合（cAR1）诱导的异三聚体G蛋白解离成Gγ和Gβγ 亚基7，9，10 。 Gβγ亚基激活Ras，这反过来又激活的PI3K，转换成11-13细胞膜上的画中画画中画3 2。作为画中画3 pleckstrin同源性（PH值）域的蛋白质结合位点，从而招募这些蛋白膜14，15 。 cAR1受体的激活还控制PTEN基因， 这 3画中画画中画2月 16日，17去磷酸化膜 ​​协会。是进化上保守的G蛋白偶联受体介导的趋化因子，如中性粒细胞18的人体细胞趋化的分子机制。我们提出了下面的方法研究D 的趋化discoideum细胞 。 1。制备趋化组件细胞。 2。成像的细胞趋化的一个营地梯度。 3。监测的异三聚体G蛋白G蛋白偶联受体在单个活细胞中的诱导活化。 4。在单个活细胞实时成像趋化触发动态画中画3反应。我们开发的成像方法可以应用于研究人类白细胞趋化作用。

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

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

在这里，我们描述了详细的调查趋化活细胞成像的方法。我们目前的荧光显微方法来监测细胞迁移的信号事件的时空动态。测量信号的事件，使我们进一步了解如何GPCR的信令网实现了梯度感应的趋化因子和控制真核细胞的定向迁移。

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 ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

Research

JoVE Journal

Biology

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

17.9K 次观看.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 在这里，我们描述了详细的调查趋化活细胞成像的方法。我们目前的荧光显微方法来监测细胞迁移的信号事件的时空动态。测量信号的事件，使我们进一步了解如何GPCR的信令网实现了梯度感应的趋化因子和控制真核细胞的定向迁移。

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

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 ...

科研

生物学

Quantifying Agonist Activity at G Protein-coupled Receptors

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 or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

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 ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

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

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 ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

调查Coronin A在早期饥饿反应中的作用<em&gt;粘菌</em&gt;通过聚集测定

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...