This work was supported in part by the National Institute on Drug Abuse T32 DA 016176, National Institute on Drug Abuse DA 038817, and National Institute on Drug Abuse DA 040047.

1.细胞培养和转染<ol><li> 维持在生长培养基小鼠神经母细胞瘤2A（的N2a）细胞。 <ol><li>使500毫升的N2a生长培养基从200毫升的Dulbecco的Eagle培养基（DMEM）与高糖250毫升降低血清培养基，将50ml胎牛血清（FBS），和5ml青霉素/链霉素（100倍）。 </li><li>保持在T75烧瓶中的细胞在37℃在一个5％CO 2培养箱。 </li><li>拆分单元格1:15在必要的时候，或者在约80-90％汇合。这通常是每周2-3次。 </li></ol></li><li> 外套35毫米玻璃底培养皿用聚-D-赖氨酸。 <ol><li>在层流的生物安全柜中，添加200微升0.1微克/毫升多聚-D-赖氨酸的到无菌35mm培养皿的玻璃底区域。 </li><li>放置培养皿在37℃培养箱中培养1小时。 </li><li>仔细冲洗菜DDH 2 O的3-4倍。 </li><li>让菜完全干燥时间超过1小时。 </li><li>与不孕IZE使用UV光生物安全柜如果有必要的菜肴。 </li></ol></li><li> 板的N2a细胞TIRFM成像。 <ol><li>从贴壁细胞中取出培养基。 </li><li>通过在5％CO 2培养箱用1ml 1X胰蛋白酶（+ EDTA）中5分钟，在37℃温育分离从烧瓶的细胞。 </li><li>加入9毫升生长介质灭活胰蛋白酶。混合用吸管媒体，胰蛋白酶，与脱落的细胞。 </li><li>目视计数使用血球细胞，并计算出正确的卷至90,000细胞添加到每个聚D-赖氨酸涂覆的玻璃底培养皿中。此密度是必需的有效转染和单细胞的TIRF成像。 </li><li>加2ml生长介质包含90000单元，每个单元玻璃底菜。 </li><li>孵育在37℃下在5％CO 2培养箱培养皿16-24小时。 </li></ol></li><li> N2A转 <ol><li>获得含有荧光SEP注册成立的质粒构建到感兴趣的蛋白质的胞外区。标准克隆技术例如PCR扩增5可以用于生成构建体。 注:SEP应纳入膜蛋白的胞外区，以便它驻留的内质网或贩卖囊泡的管腔内并暴露于细胞外溶液在质膜时。 SEP是在大小类似于GFP和类似克隆策略都可以使用。 </li><li>上镀细胞用1.5ml降低血清培养基替换生长培养基（ 例如 ，OPTI-MEM），在加入转染试剂的前约30分钟。 注:研究受体药物引起的变化，药物的适当浓度可以在转染时加入。 </li><li>将每个所需质粒的500纳克构建以250微升减少血清培养基（管1）。 </li><li>添加2微升转染试剂的一个单独的内胎Containing 250微升血清减少媒体。在室温下孵育管2 5分钟。转染试剂的质粒DNA中的比例应该被优化以表达感兴趣各蛋白质。 </li><li>结合管1和2，使含有500微升转染试剂和质粒混合的溶液。在室温下孵育25分钟。 </li><li>添加500μl的转染混合物预镀细胞为2毫升，每培养皿的总体积。 </li><li>孵育细胞在37℃下在5％CO 2培养箱24小时。 </li><li> 24小时后，除去转染混合物并漂洗与生长培养基的细胞，然后加2ml生长培养基到每个盘中。 注意:如果在转染时，加入的药物，它可以再次在该步骤补充。 </li><li>孵育细胞在37℃下在5％CO 2培养箱24小时。 </li><li>图像细胞转染后48小时。 </li></ol></li></ol>道达尔在2活细胞成像ternal反射荧光显微镜（TIRFM） <ol><li> 成像成立 <ol><li> 如图1与倒置荧光显微镜设置用扫描阶段进行摄像。这需要一个488纳米的DPSS激光源的对准，步进马达来调整488纳米的光束位置，和高数值孔径（1.49 NA）的60X或100X油浸物镜。 </li><li>通过适当的激励过滤器（带通十分之四百八十八NM）通过激光束。通过连接到安装到步进电机一个发射单模光纤对准偏振激光束。在萤光模式中，激发光束在物镜的后孔的正中。 </li><li>为了达到TIRF，使用步进电机横跨物镜的背面孔径聚焦激光束平移，直到达到临界角和光束不再透过样品和被代替全反射离玻璃 - CELl接口。 </li><li>使用具有适当的排放过滤器成像软件（ 如的Metamorph）控制的EMCCD（512×512像素）拍摄图像安装在排放路径（五十零分之五百二十五纳米）。 </li></ol></li><li> 准备影像解决方案 <ol><li>通过在DDH混合150毫摩尔NaCl，4mM的氯化钾，2mM的MgCl 2的，2毫氯化钙 ，10mM的HEPES和10mM葡萄糖制备200毫升胞外溶液（ECS）的2 O储备溶液氯化钠，氯化钾， 氯化镁 ，和氯化钙可以预先制备，并用新鲜HEPES和葡萄糖上成像的日组合。 </li><li>调整含有100ml ECS至pH 7.4的溶液的pH值。调整其余100毫升ECS的至pH 5.4。冲洗转染的细胞用2ml ECS（pH值为7.4）。 </li><li>成像前2毫升ECS液（pH 7.4）添加到转染的细胞。 </li></ol></li><li> 在全内反射荧光活细胞成像 <ol><li>将整个系统上，INCLUDIN摹488 nm激光，摄像头，移动平台和成像程序。重点落射荧光束，并在客观60X调节功率约为1毫瓦。 </li><li>添加油的目标和放置在翻译阶段转染细胞的菜。使用阶段坐骑，以确保它不会相对于移动到阶段使细胞可成像多次固定菜到位。 </li><li>在萤光模式，聚焦显微镜，并找到荧光，转染细胞。 Cells公司将继续集中在几个焦点平面。找到单一的，孤立的细胞继续进行TIRF。 </li><li>在成像程序，设置曝光时间为200毫秒，并通过设置EM增益优化荧光强度。 </li><li>通过使用步进电机平移穿过物镜的光束以步进方式转换的激光束分成TIRF。由于临界角的方法，光束将明显横跨盘的边缘，翻译，直到它收敛于次在样品平面内全反射的E点。 </li><li>验证细胞是TIRF模式通过调节调焦旋钮。在TIRF，仅在一个平面的细胞可以被聚焦（ 即约150从玻璃界面处），从而产生具有高分辨率的质膜的一个非常定义的图像。 </li></ol></li><li> 成像SEP确定的亚细胞定位 <ol><li>定位在成像平面健康，转染，单细胞。 </li><li>在pH 7.4获得细胞的聚焦图像。 SEP标记在质膜和内质网受体应该是可见的。 </li><li>迅速到达样品，以防止光漂白挡住激光束。 </li><li>记住对应使用显微镜能够记录多个XY位置的成像软件每个单元的舞台位置。 </li><li>重复步骤2.4.1-2.4.4为每皿20-30细胞，而使用软件来记录每个单元的位置。 </li><li>房颤之三，在pH 7.4的所有细胞图像收集，手动删除使用移液管将pH 7.4 ECS溶液。请勿触摸的菜，因为这可以移动存储的阶段位置。 </li><li>仔细2毫升pH值为5.4 ECS添加的菜，然后等待10分钟。在此期间，保存预先获得的图像。 </li><li>根据相同组用于在pH7.4，收集的图像，移动台到每个保存的位置和在pH 5.4获得相同的细胞的图像的条件。细胞应少看定义的，因为所有的荧光检测是从封闭SEP内质网原产标记受体。保存pH值5.4细胞图像。 </li></ol></li><li> 在活细胞成像单囊泡插入事件 <ol><li>替换转染的细胞的生长培养基与2ml的pH 7.4 ECS，或具有Leibovitz的L-15介质。 Leibovitz的L-15介质的pH为CO 2独立的，使得必要的成像发生在一段长的时间。 </li><li>的PlacE在显微镜阶段转染细胞的菜。安全和TIRF集中的单个单元格以下上面的步骤2.3。 </li><li>如果装备，设置自动对焦所以重点并不在成像期间漂移。 </li><li>记录了一系列1000帧，在200毫秒帧速率连续拍摄图像。荧光的脉冲串将在这段时间内是可见的，对应于低pH贩卖囊泡与质膜融合，露出SEP到细胞外pH为7.4。 </li><li>找到另一个单细胞，并重复上述步骤。如有必要，复位自动对焦。 </li></ol></li></ol> 3.图像分析与数据处理<ol><li> 分析SEP荧光确定的亚细胞定位 <ol><li>打开使用图像分析软件如的Metamorph或ImageJ的（http://imagej.nih.gov/ij/）的细胞的图像。减去两者的pH 5.4和pH 7.4的图像背景使用滚球设置。</li><li>使用强度基于阈值从单个细胞量化荧光。在pH值7.4图像手动选择的细胞周围感兴趣的区域。 </li><li>测量单元面积，平均强度，并使用ImageJ的插件或者使用内置的"测量"功能分析选项卡下的集成密度。 </li><li>重复步骤3.1.1-3.1.3对于相同细胞在pH 5.4，小心地调整以相同的方式兴趣的强度基于阈值和区域。 </li><li>在两个pH值7.4和5.4细胞图像获得集成密度后，通过从pH 7.4的值中减去的pH 5.4的值计算质膜集成密度。的差对应于在TIRF激发区域内的质膜的相对数量的受体。 </li><li>贩卖的量度，计算SEP的相对百分比标记位于质膜的受体相比，在TIRF excitati可见剩余受体关于通过将在pH 7.4的总集成密度集成密度质膜，乘以100卷。 </li></ol></li><li> 分析单泡插入事件 <ol><li>打开该系列产品采用图像分析软件1000 TIFF图像。减去使用滚球设置所有帧的背景。 </li><li>调整记录的色彩平衡，以最大限度地对应于囊泡插入事件激烈的区域。手动计数荧光持续时间超过3张（&gt; 600毫秒）爆发。 </li></ol></li></ol>

<ol>
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The authors have no conflicts of interest to declare.

SEP的pH敏感性使得驻留在质膜的受体从细胞内的受体的内质网进行区分，并且它可以被用来解决的插入事件受体携带囊泡5，7，8，9，18，19，20 。几种技术包括表面生物素和配体结合被广泛用于测量蛋白质表面的水平。在?...

在受体表达，分布和装配的更改已连接到各种各样的疾病，包括阿尔茨海默氏病，帕金森氏病，囊性纤维化和药物成瘾1，2，3，4，5。例如，尼古丁和其他烟碱配体的影响烟碱型乙酰胆碱受体（nAChRs），导致贩运，表达和上调1，2，5，6，7，8，9，10变化的贩卖。尼古丁增加组装nAChRs的总数的小区内，增加贩卖朝质膜，一个ð改变亚基组装偏袒某些亚型的高灵敏度版本。在疾病模型解决贩卖，组装和受体的表达明显变化提供重要的机械细节都必须确定药物靶点。一种理想的方法是细胞内受体和那些定位于质膜之间快速区分。这是在大多数特定蛋白质的驻留在细胞内，如与nAChRs的情况下尤其困难。因为大多数的nAChRs的定位于内质网，传统的测量缺乏必要查明沿分泌途径本地化和贩卖变化的时空分辨率。烟碱受体的受体贩运和表达的研究，主要是使用放射性配体结合11，生物素测定12，免疫印迹13，或免疫techniq进行<html>用户设备12。这些都依赖于细胞的报告分子或固定的结合特异性和缺乏质膜居民和胞内受体之间同时区分的能力。因此，离子通道组件和囊泡动力学的研究已经在很大程度上依赖于低通量电生理技术14。 优越的空间和时间分辨率可以在荧光显微镜的进展。遗传编码的报道分子，如绿色荧光蛋白（GFP）及其变体，消除非特异性结合的问题，并提高灵敏度15。 GFP的pH敏感的变体，被称为superecliptic pHluorin（SEP），可以被用来利用在小区内室之间固有的pH差异来确定定位5，7，8，裁判"&gt; 9，16，17，18。SEP荧光当pH高于6，但在较低pH保持在关断状态，因此，在其腔侧具有标记SEP受体检测存在于内质网时（ ER）或在插入质膜（PM），而不是当局限于一个贩卖囊泡。细胞外pH值在与质膜受体接触的操纵从而改变了荧光，因此，检测这些受体的，如果在同一小区在两个中性外pH，然后pH值低于6顺序地成像，所述图像之间的差异归因于位于质膜的受体。这使得细胞内的一个同时测量（外围ER）和质膜驻地受体5， 7,8 </suP&gt; 9。单泡囊插入事件，也可以当细胞外pH为中性解决。一旦低pH贩卖囊泡与质膜融合，所述囊泡的腔侧暴露于中性细胞外溶液，导致检测到的荧光7，18，19，20的突发的过渡。 SEP使定位于质膜和外围内质网的受体的测量，并提供测量这些亚细胞区5，7，18之间受体贩卖的装置。 以实现在质膜更高的分辨率，与SEP受体基因编码的是通过全内反射荧光显微镜（TIRFM）成像。这种方法是特别有用的，如果大多数受体定位于细胞内的区域中，由于TIRFM增加质膜的可视性。 TIRFM还支持单泡携带在插入SEP标记的受体进入PM贩运动态的分辨率。全内反射发生在具有不同折射率，例如作为细胞和玻璃盖玻片21，22之间的材料的界面。当与488 nm激发，其被定向以实现在玻璃和细胞溶液的界面的全内反射照射SEP荧光。这将产生约穿透150纳米到样品，本卷中唯一令人振奋的荧光衰减波。仅检测含有在上述范围内的激发内中性pH环境受体SEP，对应于那些居住在质膜或外周的内质网。由于检测是有限的牛逼通过渐逝波，从细胞内区域的背景荧光ö激发减小并且信噪比升高21,22。此外，由于辐射不穿过本体单元的，光损伤被最小化，允许在一定时间的过程中活细胞成像。其结果是，TIRFM加上遗传编码SEP提供了测量的膜受体的亚细胞定位和贩卖动力学沿分泌途径所需的高的分辨率和灵敏度。

<table><tbody><tr><td>&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Flasks with Filter Cap, Sterile, Greiner Bio One, 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>VWR</td><td>82050-856</td><td></td></tr><tr><td>35 mm glass bottom Petri dishes, sterile</td><td>Cell E&amp;amp;G</td><td>GBD00002-200</td><td></td></tr><tr><td>Poly-D-lysine</td><td>vwr</td><td>215017510</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium&amp;lrm; (DMEM), High Glucose</td><td>Fisher Scientific</td><td>11-965-084&amp;nbsp;</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco / Fisher Scientific</td><td>31-985-088</td><td></td></tr><tr><td>Fetal Bovine Serum, Certified, US Origin, Standard (Sterile-Filtered)&amp;nbsp;</td><td>Gibco / Fisher Scientific</td><td>16-000-044</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), no phenol red</td><td>Fisher Scientific</td><td>12604-021</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>VWR</td><td>45000-652</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco / Fisher Scientific</td><td>21083027</td><td>Optional</td></tr><tr><td>Lipofectamine</td><td>Fisher Scientific</td><td>11668030</td><td>Gently mix; Do not vortex</td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-10</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Objective immersion oil&amp;nbsp;</td><td>Olympus</td><td>Type F</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>Camera</td><td>Andor</td><td>iXon Ultra 897</td><td></td></tr><tr><td>60X, 1.49 NA oil immersion objective</td><td>Olympus</td><td>APON 60XOTIRF</td><td></td></tr><tr><td>Motorized stage</td><td>Prior</td><td>IXPROXY</td><td></td></tr><tr><td>Motorized actuator (stepper motor)</td><td>Thorlabs</td><td>ZST213</td><td></td></tr><tr><td>MetaMorph (or other imaging program)</td><td>Metamorph</td><td></td><td></td></tr><tr><td>488 nm laser</td><td>Market Tech</td><td></td><td></td></tr><tr><td>Single mode fiber</td><td>Thorlabs</td><td>SM450</td><td></td></tr><tr><td>Mirrors</td><td>Thorlabs</td><td>BB1-E01</td><td></td></tr><tr><td>Dichroic 488 nm LP</td><td>Semrock</td><td>Di02-R488-25x36</td><td></td></tr><tr><td>Bandpass filter, 488 nm</td><td>Semrock</td><td>LL01-488-12.5</td><td></td></tr><tr><td>Bandpass filter, 525/50</td><td>Semrock</td><td>FF03-525/50-25</td><td></td></tr></tbody></table>

utilizing phluorin-tagged receptors to monitor subcellular localization and trafficking

了解膜蛋白运输，组装和表达要求那些居住在细胞器以及定位于细胞膜区分的方法。传统的基于荧光的测定缺乏区分驻留在不同细胞器的膜蛋白的能力。切削刃方法超越通过用全内反射荧光显微镜（TIRFM）偶联pH敏感的荧光团的传统方法。 TIRF照明激发样本以将其从玻璃样品接口约150纳米，从而降低背景，该信号增加信噪比，并提高分辨率。在TIRFM激发体积包含质膜和附近的细胞器，如外围ER。 Superecliptic pHluorin（SEP）是GFP的pH敏感的版本。遗传编码SEP到感兴趣膜蛋白的胞外域位置上的荧光团对ER的和在细胞的胞外区的腔侧。 SEP是荧光当pH大于6，但在较低pH值保持在关断状态。因此，受体在质膜（PM）驻留在内质网（ER）或在插入时而不时局限于一个贩卖囊泡或其它细胞器如高尔基具有标记SEP发出荧光。细胞外pH值可以被调节以决定受体的荧光上质膜。在TIRF图像之间的荧光在中性和酸性外pH为同一小区中的差对应于在质膜受体的相对数。这使得细胞内和细胞质膜驻地受体的同时测量。单泡囊插入事件，也可以当细胞外pH值是中性的，对应于低pH贩卖囊泡与质膜融合，并转换成荧光状态测量。这的VersatilË技术可以被利用来研究定位，表达和膜蛋白的贩卖。

SEP纳入受体允许在活细胞直接检测的受体。再加TIRFM，这允许在质膜和受体在TIRF激发区域中的亚细胞位置中的分布的相对表达水平的评估。单囊泡运输事件也可以解决。 在质膜SEP标记的受体相对表达水平激发后SEP荧光被周围溶液的pH值所决定的。当SEP与质膜驻地受体融合，细胞外pH?...

Watch this Scientific Journal Video about Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking at JoVE.com

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

标签膜蛋白的胞外结构域与pH敏感荧光，superecliptic pHluorin（SEP），允许确定的亚细胞定位，表达和贩卖。成像SEP-标记的蛋白质与全内反射荧光显微镜（TIRFM）使蛋白质水平在外围ER和质膜的量化。

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 ...

utilizing-phluorin-tagged-receptors-to-monitor-subcellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.7K 次观看.  University of Kentucky. 标签膜蛋白的胞外结构域与pH敏感荧光，superecliptic pHluorin（SEP），允许确定的亚细胞定位，表达和贩卖。成像SEP-标记的蛋白质与全内反射荧光显微镜（TIRFM）使蛋白质水平在外围ER和质膜的量化。 

视频: Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

科研

神经科学

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 ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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 ...

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 ...

生物学

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

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 ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...