Name: visualization of endoplasmic reticulum subdomains in cultured cells
Uploaded: 2014-02-18T13:45:00
Description: Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells at JoVE.com

作者感谢基金会Filarete提供的帮助和支持，在此文章的发表。我们还要感谢炫酷那么Europeo二Nanomedicina为使用该Tecnai G2透射电子显微镜。

1。质粒，细胞培养，转染，并与ER荧光蛋白<ol><li>在本研究中使用的质粒由GFP的增强版本融合在其C-末端的尾部区域的大鼠色素b的ER亚型（5）通过接头序列（如B（5）此处略）。尾部区域包含整个序列（Pro94-Asp134），该保持天然B的胰蛋白酶切割位（5），其中包括17个残基的TMD（跨膜结构域），由上游和下游的极性序列侧接（UPS和DPS）后膜相关。连接器包括myc表后跟[（甘氨酸）4辑] 3?...

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本文所描述的协议和成像方法已被用于研究转助教荧光蛋白居民在活细胞的内质网的分布和流动。我们也分析了这个亚细胞区室中通过超微结构分析装置的体系结构中的过度表达，这些蛋白质的效果。 活细胞共聚焦成像和电子显微镜的组合代表是一个非常强大的研究蛋白质的动态性能的手段，并可能提供有关蛋白质功能的重要信息。所描述的方法不是费时（一般工作三天）?...

绿色荧光蛋白（GFP）及其变种光谱和荧光显微镜的并行开发的发现，开辟了全新的途径，在细胞中蛋白质的行为进行调查。技术，如荧光漂白恢复（FRAP）和荧光损失漂白（FLIP），这是因为荧光的内在能力，以扑灭在强光照射下其荧光的可能后，基于共聚焦活细胞成像和使用的转荧光融合蛋白1-3。它们被广泛用于评估蛋白质不仅是本地化，而 ​​且他们的流动性和膜泡运输，它可以揭示有关它们的功能4重要线索。 真核细胞的独特之处在于具有特定的脂质和蛋白组成的细胞内隔室的存在。虽然细胞器是物理隔离器版，它们需要彼此通信，并以维持细胞稳态交流分子成分。分泌途径保证了在ER中合成的蛋白质和脂质到达正确的最终目标，其中它们发挥它们的功能。细胞内细胞器，也可以通过动态接触部位，使分子（脂质）向隔间之间直接交换连接。此外，许多蛋白已到组装在大异聚复合物，或与特定的脂类物质（脂质筏/微区）为了成为功能活性或者被运送到最终目的地相关联。所有这些生物方面极大地影响蛋白质的动力学性质，并因此可以通过如下所述的技术手段进行适当的调查。 我们的团队已经广泛应用于FRAP和FLIP结合电子显微镜来研究内质网的结构和它的不同的子域。内质网是分泌途径的第一站和在蛋白质和脂质排序5起关键作用。这是一个高度动态的细胞器，其独特的子域名反映其许多不同的功能（ 如蛋白质和脂质的合成和贩运，蛋白质折叠，Ca 2 +的储存和释放，以及异物代谢）。然而，尽管它们在形态上，空间上，和功能上不同，这些结构域彼此连续，并且它们的相对丰度可在细胞生理和病理条件下进行修改。最有名的和内质网中，通常在空间上分离的结构域是核膜和平滑和粗糙的ER，但是，我们和其他人已经证明，有ER结构具有更复杂的结构和三维组织在各种细胞类型和在生理条件下的组织，也可以由紧张刺激如缺氧，药物诱导的手段管理或过度表达的ER驻留跨膜蛋白2,6（以及其中的参考文献）。 我们最近还展示了这样的结构在人类疾病中1,7的细胞模型的存在。从滑面内质的堆积潴发起，他们分别获得组织光面内质网（OSER）的总称，2003年6，虽然他们也被称为karmellae，片状结晶 ，并且晶体ER他们的架构的基础上，像他们的大小，可以改变。之后，细胞转染绿色荧光蛋白融合到尾锚（TA）ER居民蛋白质（D EGFP-ER），绿色荧光蛋白在转弱二聚化倾向显着地改变了ER的组织和结构的细胞质区域。 FRAP和FLIP实验表明使得 d EGFP-ER是自由OSERs内扩散，并且它从网状ER移动到OSER 反之亦然&lt;事实/ em&gt;的指示聚集是连续的与周围的网状急诊室。超微结构分析使我们能够将荧光数据与OSER体系结构和组织在纳米尺度水平上的详细描述关联：OSERs总是由成堆的光滑ER配对囊泡的，但可能有不同形式的空间组织，如规则排列的正弦数组或轮生，或六角形“晶体”管阵列。这些重排导致立方形态8的，因为它们在细胞中发现的生理条件9和下面的压力，如缺氧10，药物治疗11和癌症9下，可有显 ​​著电位超微结构标记。 使用GFP融合蛋白第一次演示之后，我们使用了成像实验，分析ER结构域的增殖响应于药物治疗12，ASSESS荧光蛋白的倾向oligomerise在细胞13，并调查的突变，ALS联TA蛋白在ER起源的细胞内聚集，可能是有关其致病1,8的形成中的作用。它已被提出，形成的细胞内聚集物（这发生在许多神经退行性疾病14）可以设计成防止有毒的突变蛋白和周围的细胞成分15之间的相互作用的一种保护机制。 以下是对光学和电子显微镜方法考察构建体的C-末端的疏水性结构域被插入到内质网的膜，并且其动态行为的分析以及其过度表达对ER域的效应的组合的说明在培养的细胞结构（参见图1的实验方案的流程图）。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>41966029</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle medium (DMEM) w/o phenol red</td>
			<td>Invitrogen</td>
			<td>31053028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-Glutamine 200 mM solution</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		 </tr>
		 <tr>
			<td>jetPEI</td>
			<td>Polyplus Transfection</td>
			<td>PP10110</td>
			<td></td>
		 </tr>
		 <tr>
			<td>OxyFluor</td>
			<td>Oxyrase Inc.</td>
			<td>OF-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde Grade I</td>
			<td>Sigma Aldrich</td>
			<td>G5882</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Cacodylate Trihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Osmium Tetroxide 4% solution</td>
			<td>Electron Microscopy Science</td>
			<td>19150</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Uranyl Acetate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>73943</td>
			<td>slightly radioactive</td>
		 </tr>
		 <tr>
			<td>Propylene Oxide</td>
			<td>Sigma-Aldrich</td>
			<td>82320</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EPON embedding medium kit</td>
			<td>Sigma-Aldrich</td>
			<td>45359-1EA-F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lead Citrate </td>
			<td>Electron Microscopy Science</td>
			<td>17800</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bench top centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectral Confocal Microscope </td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CO2 Microscope Cage Incubation System </td>
			<td>OkoLab</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ultramicrotome </td>
			<td>Leica Microsystems</td>
			<td>UC6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Diamond knife </td>
			<td>Diatome</td>
			<td>Ultra 45 &deg;</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmission Electron Microscope </td>
			<td>FEI</td>
			<td>Tecnai G2</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GraphPad Prism Software</td>
			<td>GraphPad Software, Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>steel culture cell chamber for 24 mm coverslip</td>
			<td>Bioscience Tools</td>
			<td>CSC-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Electron Microscopy grids</td>
			<td>Electron Microscopy Science</td>
			<td>G300Cu</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

visualization of endoplasmic reticulum subdomains in cultured cells

在真核细胞中的脂质和蛋白质细胞区室之间不断交换，但这些保留其独特的组成和功能，尽管激烈的interorganelle分子的流量。在本文中描述的技术是强大的研究蛋白质和脂质的流动性和贩运体内 ，并在其生理环境的手段。光漂白（FRAP）和荧光损失在光漂白（FLIP）后荧光恢复被广泛用于活细胞成像技术通过外 - 内吞途径，细胞器或子隔离之间的连续性，形成蛋白质复合物，以及蛋白质定位研究细胞内运输所有这些脂质微区，可生理和病理条件下观察。这些方法的局限性主要是由于使用了荧光融合蛋白，以及它们的潜在缺点包括伪迹过表达离子在细胞内和在标记的和天然蛋白质的折叠和定位的差异的可能性。最后，由于光学显微镜（约200纳米）的分辨率的极限不允许急诊室或特定的小班，可以在细胞应激状态下起源的精细结构的研究（ 即缺氧，药品监督管理，过度表达的跨膜ER常驻蛋白）或病理条件下，我们结合培养的转染细胞的活细胞成像与超微结构分析了基于透射电子显微镜。

图2显示了蛋白质的流动性的一个例子FRAP研究。 ðEGFP-ER蛋白的迁移率证明了在漂白漂白OSERs后迅速荧光恢复。对于定量分析，在中场休息时和移动部分均来自实验测得的数据进行拟合以下单指数方程： F（T）= F + 后 （F REC-F后 ）（1-E-T /τ） 其中F是后荧光信号漂白后，F REC</sub...

Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells at JoVE.com

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

我们描述了使用调查转染荧光蛋白通过活细胞的共聚焦成像装置驻留在内质网（ER）的分布和流动的成像方法。我们还超微结构分析其表达对这个亚细胞区室的结构的效果。

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生物学

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Chromosome Preparation From Cultured Cells

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞：S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...