Name: tracking drug-induced changes in receptor post-internalization trafficking by colocalizational analysis
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这项工作是由CIHR（MOP394808）和加拿大研究主席CMCEWO资助项目是由NSERC的研究生奖学金的获得者。

注:此协议是与各种单层镀细胞/组织培养模型，药物治疗方案，并标注大致目标兼容。因此，在实际使用中，许多具体的参数将根据实验设计而有所不同。这里，引用这些用户定义的参数都是通用的。实施例的条件下，作为用于获得代表性的结果，被包括在斜体。 1.解决方案<ol><li>制备洗涤缓冲液通过混合0.1M Tris缓冲高渗（300毫摩尔）盐水和0.05％的吐温20; pH为7.4，在室温。 </li><li>准备封闭液。到的0.05M Tris缓冲高渗（300毫）盐水加0.05％的吐温20,3％牛血清白蛋白（BSA）和0.1％冷的鱼明胶的皮肤和将pH调节至7.4，在室温。 </li><li>制备抗体稀释液中加入0.05％的吐温20,1％BSA，0.1％冷的鱼明胶的皮肤到0.05M的Tris缓冲高渗（300毫摩尔）盐水和将pH调节至7.4，在4℃。 注意:Tris缓冲液的pH值是温度依赖性的。即，在温度变化将改变溶液的pH。为了保持一致性，这些缓冲器应该总是pH调节在它们将被用在其中温度。 </li></ol> 2.细胞培养注意:根据所用细胞类型合适的细胞培养协议将变化。这些程序必须单独优化。详细的细胞培养方法都一应俱全，其中包括16个 。类似的方法用于获得代表性的结果，与显着的差异: <ol><li>从成年动物文化背根神经节的神经元所描述的12。使用成人神经细胞生长培养基来培养细胞。减少神经胶质细胞密度由预镀，而不是谷氨酸。这导致生长在12轮的玻璃盖玻片每板至少有30-60元和相应的神经胶质细胞的混合神经元 - 胶质细胞培养中生长到approximat伊利40％汇合。应当指出的是，在原代培养的神经元将不会复制并通过共培养的神经胶质细胞被推离该板的，如果神经胶质允许过度生长。为了避免影响的神经元，神经胶质数量的物理还原优选化学/药理学方法。 注:在本协议的目的，我们假设感兴趣的细胞生长到他们的实验，理想状态和单层镀。我们发现，在24孔板上镀敷12轮的玻璃盖玻片是最方便的。所有卷和程序描述了适合于在24孔板的一个孔1盖玻片。 </li></ol>培养细胞3.药物治疗注意:多个顺序的，或重叠，药物治疗是可能的。的药物，剂量/浓度，并用于将依赖于特定的实验曝光的持续时间。 <ol><li>取出原有的生长培养基16 </SUP&gt;与含有感兴趣的药物（多个）在所选定的浓度（S）培养基更换。在这种情况下，使用10μM的吗啡溶解在盐水中，或使用盐水作为对照。 注意:重​​要的是，每个独立的复制（一般为单24孔板培养物）含有相同的公共控制条件。这将允许数据在重复正常化，这对于纠正审间的差异。 </li><li>孵育在原生长条件为48小时16细胞。 </li><li>对于后续药物治疗，重复步骤3.1和3.2与δ啡肽1微米，SNC80 1微米，或车辆，并培育60分钟12。溶解δ啡肽II在水和在40μM的HCl和超声，在水中稀释至1mM溶解SNC80以10mM。 注意:此协议假定药物（多个）已被溶解，使得它们可在在所选择的生长培养基中的所需浓度（多个）中溶解。适当程序这种增溶将取决于所讨论的药物而变化，并且必须单独优化。 </li><li>轻轻洗细胞三次，以约1毫升每次洗涤的洗涤缓冲液。通过从井轻轻抽吸液体，然后迅速，并轻轻地，再填充井用新鲜溶液中，根据需要（在这种情况下，用洗涤缓冲液）进行每次洗涤。 </li></ol> 4.固定和免疫组化注意:特定标记目标将通过实验而变化。在我们的使用中，我们标记三角洲阿片受体（DOR）和Rab5的，拉布11，和LAMP1，为讨论。所述特异性抗体用于将取决于具体的实验。最佳标记条件是依赖于特定的抗体使用（多个）。这个话题的更全面的讨论如下。 <ol><li>固定在〜细胞通过浸没在0.1M磷酸盐缓冲盐水将300μl的4％多聚甲醛在37ºC10分钟。 注意:重​​要的是，在4％的对甲醛被新鲜由于因使用先前冷冻的多聚甲醛的自体荧光制备。 </li><li>轻轻洗细胞3次，以约1毫升每洗清洗缓冲液，如在3.3中描述。 </li><li>孵育细胞中加入300微升封闭缓冲液中进行2小时，在室温。 </li><li>孵育细胞在300μl的抗体稀释液进行48小时的初级抗体在4ºC。在我们的使用中，对DOR初级抗体（兔抗DOR），并之一:Rab5的（小鼠抗Rab5的），拉布11（鼠抗Rab11），和LAMP1（羊抗LAMP1）。 </li><li>轻轻洗细胞三次，以约1毫升每洗清洗缓冲液，如在3.3中描述。 </li><li>孵育细胞与缀合到不同的荧光第二抗体（见下面的讨论）在300微升抗体稀释剂进行1小时，在室温。在此，所有的后续步骤，保护细胞免受光。以获得的代表性结果，使用绿色发光荧光团缀合的抗兔和eith呃发射红色光的荧光团缀合的抗 - 小鼠或红色发光荧光团缀合的抗山羊。 </li><li>轻轻洗细胞3次，以约1毫升每洗清洗缓冲液，如在3.3中描述。 </li><li>留下约1毫升洗涤液，每孔，端着盘子在45°〜取下24孔板12轮的盖玻片，并撬动盖玻片关井地面用细镊子。盖玻片会靠在孔壁，从那里它可以容易地使用镊子除去。 </li><li>安装盖玻片，细胞面朝下，在显微镜载玻片与抗褪色封固剂。 </li></ol> 5.显微镜设置<ol><li>使用高放大倍数的物镜（100X），首先找到一个代表性的细胞，以萤光使用成像。 </li><li>配置图像采集设置，记录每一个标记通道8位未压缩的TIFF图像（用于检测每个例如荧光团，488 nm和594纳米），将图像的每一个通道顺序地（或者通过线或帧），并以平均4至6的扫描为最终的图像。最佳的图像分辨率将显微镜专用高，但1024×1024通常会产生很好的效果。 </li><li>切换到共焦成像路径，并通过单元的中心聚焦到Z-平面。 </li><li>优化针孔（通常为1艾里单位），激光功率和光电倍增管电压（增益），并为每个频道偏移。记录这些设置保存/。使用相同的显微镜设置用于成像的所有标记的​​针对任何给定目标对在同一复制的细胞。 注意:此过程通常会导致足够漂白该单元不应该被成像进行分析。 </li></ol> 6.成像<ol><li>使用高倍率的目标（ 如 100X），首先定位一个小区使用萤光成像。 </li><li>切换到共焦成像路径，并通过单元的中心聚焦到Z-平面。一世˚F可用，使用软件缩放/作物以限制扫描区域对感兴趣的细胞。 </li><li>拍摄图像使用的设置每个通道标记以前建立的。重复步骤6.1至6.3，以图像每复制条件15个或更多的细胞，以确保有足够的样本。 </li></ol> 7.共定位分析<ol><li>打开对图像（ 例如 ，"绿色"和"红"）中的ImageJ一个细胞。使用图像&gt;颜色&gt;合并通道...命令生成一个RGB图像。 </li><li>绘制所述感兴趣的细胞周围的选择。使用ImageJ的插件"PSC共定位"（11;可从https://www.cpib.ac.uk/tools-resources/software/psc-colocalization-plugin/），以量化的指标共存的选定单元格中。 </li><li>录制所需共存措施（通常皮尔森R 17）。重复步骤7.1至7.3的每个细胞成像。 </li></ol&gt; 8.数据标准化<ol><li>计算的每个公共控制条件的记录，共定位值的平均值复制的每个标记条件。 注意:例如，神经元中的共定位值的平均值"48小时车辆，60分钟车"中的每一个在每3个标记条件（受体和每3个隔间）3次独立的重复药物条件。 </li><li>相乘的装置由（-1），得到的偏移为每个复制在每个标记条件。添加偏移每个复制在每个标签的条件，在每个重复值共存。 注意:这将正常化的数据，使得平均共定位措施的共同控制条件是0中的每一个标记条件的每个复制。 </li><li>从每个标签条件的所有重复池中的数据。 注:在共存现在可以在重复进行分析。统计肛门ysis将取决于实验设计，但将典型地涉及分析-的方差（ANOVA），随后进行适当的事后测试（ 例如 ，图基）。对于统计资源， 如见，18。 </li></ol>

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The authors have nothing to disclose.

我们已经优化了这个协议，成人背根神经节神经元（初级感觉神经元）的原代培养的分析。它也可以使用，很少或根本没有修改，为单层镀培养细胞广泛。该colocalizational分析也有可能在组织切片和其他此类制剂11，但药物治疗和组织固定/制备组分是不恰当的。 有趣的是，这里介绍的IC卡的方法也可以使用，以适当的固定和组织准备，以便在组织切片进行双标记免疫组...

受体，尤其是G蛋白偶联受体（GPCR），例行贩卖细胞内，并从细胞表面1。这些复杂地策划和严格控制的流程决定细胞的受体提供补充和调节受体活性的时间，脱敏和复敏2 - 4。重要的是，这些方法是响应于蜂窝环境中，包括药物诱导的受体活性或不活动。也就是说，配体在受体的行动可以改变这些受体的细胞内转运，从而改变细胞的反应。以这种方式，外部的配体在细胞功能的更多的效果发挥的是，甚至超出古典信使到效应器级联5,6。 审视这种变化引起的受体运输是困难的。所有可用的技术包括限制。生物素保护测定法已被用于MONI器表面受体的人群。这些受体的生物素化和免疫沉淀的时间过程被执行以量化在一段时间内的生物素化的受体的减少。这种技术基本上是监视一个初始的，标记的人口受体7的逐渐退化，并且是在构建这个过程的时间进程非常有用的。不幸的是，该测定是无法监测任何比受体如内化，回收或新受体的原始池的劣化等过程。此外，除了在150kDa范围在的50kDa范围内的受体的抗体的可改变该受体的贩卖8,9，并且该技术可能难以与低表达级别受体使用。 其他程序使用各种方法来确定细胞内运输隔室（ 例如，内体等），并且评估其共定位与感兴趣的受体。这包括了我们Ë异源表达系统的荧光蛋白标记的受体和隔间的标记（ 如 RAB-GTP酶家族）的嵌合结构。这潜在地允许使用活细胞成像，去除与固定和透化的问题。虽然功能强大，这样的策略，从异源系统中一般的相同的限制患有:标记和表达水平的影响对贩卖行为和不兼容的多种生理代表性的细胞类型。更普遍，染料用于轻松标记细胞内间隔（ 如溶酶体，表面上）10。染料，但是，可以缺乏特异性（染料溶酶体的情况下，所有的酸性细胞器），不要通过其他车厢评估贩卖。尽管如此，这些技术允许相当大的控制系统和实验条件，并且可以从这里提出，下面的共定位的分析方法中受益。 该方法W¯¯E存在这里由共存提炼受体贩卖的跟踪。使用免疫细胞化学（ICC）来标记适当的标记，可以识别多个不同的细胞内隔室中。这也允许在代替异源系统的使用生理学上相关的原代细胞培养的。这ICC协议涉及到固定之前标记感兴趣的细胞;这允许标签在下列药物治疗（多个）特定时间点。这将产生全球性受体室协会"快照"在那个时间点。随着多个时间点，贩运变化也时间过程可以构造。 简言之，将细胞是药物处理，标记对受体和感兴趣胞内区室，共焦成像，并且显微照片进行分析，以数学量化受体和隔室11的共定位。在我们的使用中，我们研究了与镭受体的共定位B5，Rab11，和溶酶体相关膜蛋白1（LAMP1）。这些标记识别早期内涵体，回收内涵体，和溶酶体，分别。这些措施共存作为代理人的内化，再利用和降解12的总体进程。 如同所有的技术，一些限制应该被考虑。由于需要将图像的每一个个体的神经元分析，该技术会变得非常劳动密集取决于所涉及的条件和时间点的数量。所有免疫标记也必须与所造成的固定和通透13细胞超微结构，蛋白定位和表位的辅助作用。 虽然用于与初级感觉神经元的原代培养用原本优化，这种方法与其他单层镀培养模型广泛兼容。 使用数学量化测量电Ë共定位是，值得注意的是，更严格的方法上比用来评估受体转运的变化，这往往依赖于模糊的，主观的措施，如视觉检查，多渠道覆盖14以前的技 ​​术。 这种技术对于其与体内干预（前原代培养物代）广泛的兼容性特别有用的， 在体外的干预（培养物生长期间），以及各种标记的目标15。因此，它可以适合于许多不同的研究问题。

<table><tbody><tr><td>Trizma Base</td><td>Sigma Aldrich</td><td>T1503-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S9888-500G</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma Aldrich</td><td>258148</td><td></td></tr><tr><td>Albumin from Bovine Serum</td><td>Sigma Aldrich</td><td>A7906-100G</td><td></td></tr><tr><td>Gelatin from cold water fish skin</td><td>Sigma Aldrich</td><td>G7041-100G</td><td></td></tr><tr><td>Corning Costar Cell Culture Plates: 24-well</td><td>Fisher Scientific</td><td>720084</td><td></td></tr><tr><td>12 circle Microscope Cover Glass</td><td>Fisher Scientific</td><td>1254580</td><td></td></tr><tr><td>Aqua/Poly-Mount</td><td>Polysciences</td><td>18606-20</td><td></td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Aldrich</td><td>S9638-500G</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>S9763-500G</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Polysciences</td><td>00380-1</td><td></td></tr><tr><td>Dumont #5 Forceps - Standard/Dumoxel</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Rabbit anti-DOR antibody</td><td>MyBioSource</td><td>MBS316175</td><td>Used at 1:1,500</td></tr><tr><td>Mouse anti-Rab5 antibody</td><td>Sigma Aldrich</td><td>R7904</td><td>Used at 1:750</td></tr><tr><td>Mouse anti-Rab11 antibody</td><td>Millipore</td><td>05-853</td><td>Used at 1:500</td></tr><tr><td>Goat anti-LAMP1 antibody</td><td>Santa Cruz</td><td>SC8098</td><td>Used at 1:750</td></tr><tr><td>Donkey anti-rabbit Alexa 488 conjugated antibody</td><td>Life Technologies</td><td>A-21206</td><td>Used at 1:200 to 1:2,000</td></tr><tr><td>Goat anti-mouse Alexa 594 conjugated antibody</td><td>Life Technologies</td><td>A-11005</td><td>Used at 1:200 to 1:2,000</td></tr><tr><td>Donkey anti-goat Alexa 594 conjugated antibody</td><td>Life Technologies</td><td>A-11058</td><td>Used at 1:200 to 1:2,000</td></tr></tbody></table>

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.

使用这种技术，有可能对量化以下二者慢性/长期和急性药物治疗在受体后的内化贩卖的变化。后的药物治疗，固定，和标签，高分辨率双通道显微照片被捕获的每个感兴趣细胞。代表图像可与假色共定位组合以生成所说明的图（ 图1）。随后colocalizational分析，如上所述，得到目标靶共定位（ 例如，受体隔室标志物）的定量分数。这些数据然后可用于比较的变化，在这种情况下...

Watch this Scientific Journal Video about Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis at JoVE.com

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

受体运输调节信号传导和细胞反应的配体和是，本身，响应于细胞的条件，包括配体诱导的信号传导。在这里，我们描述了一个强大而灵活的技术评估定量使用免疫标记和colocalizational分析药物诱导的受体贩卖。

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 ...

tracking-drug-induced-changes-receptor-post-internalization

Research

JoVE Journal

Biology

8.7K 次观看.  University of California Irvine. 受体运输调节信号传导和细胞反应的配体和是，本身，响应于细胞的条件，包括配体诱导的信号传导。在这里，我们描述了一个强大而灵活的技术评估定量使用免疫标记和colocalizational分析药物诱导的受体贩卖。 

视频: Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

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., ...

科研

生物工程

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

生物化学

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

神经科学

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

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.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

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 ...