Name: utilizing phluorin-tagged receptors to monitor subcellular localization and trafficking
Uploaded: 2017-03-16T14:00:00
Description: Watch this Scientific Journal Video about Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking at JoVE.com

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。 </l...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="861">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Reagent/Material</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Flasks with Filter Cap, Sterile, Greiner Bio One, 75 cm^2</td>
			<td>VWR</td>
			<td>82050-856</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:413px;">35 mm glass bottom petri dishes, sterile</td>
			<td style="width:163px;">Cell E&#38;G</td>
			<td style="width:119px;">GBD00002-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Poly-D-lysine</td>
			<td style="width:163px;">vwr</td>
			<td align="right">215017510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco Modified Eagle Medium&#8206; (DMEM), High Glucose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>11-965-084&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td>31-985-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fetal Bovine Serum, Certified, US Origin, Standard (Sterile-Filtered)&#160;</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td>16-000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express Enzyme (1X), no phenol red</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">12604-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin Solution</td>
			<td>VWR</td>
			<td>45000-652</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:413px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td style="width:119px;">21083027</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Lipofectamine</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">11668030</td>
			<td>Gently mix; Do not vortex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>P217-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective immersion oil&#160;</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:119px;">Type F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Name</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Equipment</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Microscope</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:119px;">IX81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Camera</td>
			<td style="width:163px;">Andor</td>
			<td>iXon Ultra 897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">60x, 1.49 NA oil immersion objective</td>
			<td style="width:163px;">Olympus</td>
			<td>APON 60XOTIRF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Motorized stage</td>
			<td style="width:163px;">Prior</td>
			<td>IXPROXY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Motorized actuator (stepper motor)</td>
			<td style="width:163px;">Thorlabs</td>
			<td>ZST213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">MetaMorph (or other imaging program)</td>
			<td style="width:163px;">Metamorph</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">488 nm laser</td>
			<td style="width:163px;">Market Tech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Single mode fiber</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:119px;">SM450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Mirrors</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:119px;">BB1-E01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dichroic 488 nm LP</td>
			<td style="width:163px;">Semrock</td>
			<td>Di02-R488-25x36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Bandpass filter, 488 nm</td>
			<td style="width:163px;">Semrock</td>
			<td>LL01-488-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Bandpass filter, 525/50</td>
			<td style="width:163px;">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 ...

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

细胞的细胞大分子运输检测在植物利用基因枪法

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

科研

生物学

Gastrointestinal Motility Monitor (GIMM)

Gastrointestinal Motility Monitor GIMM

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

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

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 ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

SCAP分析<em&gt;ñ</em&gt; -glycosylation和贩卖人体细胞

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

真菌细胞的活细胞成像研究抗真菌植物防御的进入和亚单位定位

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...