Name: study of the actin cytoskeleton in live endothelial cells expressing gfp-actin
Uploaded: 2011-11-18T13:00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

GFP -β-肌动蛋白的质粒慷慨提供韦恩奥尔博士，LSUHSC - S病理科，并在实验室贝基Worthylake博士，药理学教研室LSUHSC - NO扩增。这项工作是支持的国立卫生研究院（P20 RR - 018766）和美国心脏协会（05835386N）的赠款。

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GFP -肌动蛋白在活的内皮细胞成像使炎症刺激的反应的肌动蛋白细胞骨架的动态进行详细的分析。这种方法也可能是有益的借鉴以往的研究结果显示在细胞骨架重塑反应的物质力量，如剪应力 11 。此外，此方法允许的贡献的内皮细胞的活动，包括迁移，有丝分裂，形成间的路口和交界成熟，和屏障功能的维护的肌动蛋白细胞骨架动力学的详细评估。 在显示的数据，内皮细胞肌动蛋白细胞骨架的行为，可以观察治疗前后凝血酶。一直内皮细胞边缘的地方lamellipodia，观察nonconfluent和融合细胞单层形成，并随着时间的推移倒退。与凝血酶治疗短暂地打断lamellipodia形式ATION和营业额。凝血酶也引起细胞略有合同，与以前的报告中，凝血酶导致肌动蛋白应力纤维在12-14内皮细胞的形成和增加向心力紧张发展一致。然而，像这样的活细胞成像研究，现在能确定的应力纤维的起源。在HUVEC，大部分的应力纤维起源于细胞外围，类似横弧纤维细胞迁移8，9。这种方法比使用固定细胞的另一个优势是在单个细胞应力纤维的数量可以量化前后凝血酶治疗，消除选择实验组之间的偏见。 有了这个协议，我们评估在细胞边缘和肌动蛋白应力纤维的动态运动。要了解漂白后的内皮细胞肌动蛋白单体的动态，更先进的技术，如荧光恢复（FRAP）或荧光斑点显微镜复制（密克罗尼西亚）可用于15，16。此外，由于微血管内皮细胞可能代表了一种更好的模型微血管屏障功能，优化转染协议，有效地表达GFP -肌动蛋白在微血管内皮细胞代表一个逻辑的未来发展方向。 总之，生活的内皮细胞表达GFP -肌动蛋白的成像代表一个强大的工具，以确定如何内皮细胞肌动蛋白细胞骨架响应各类刺激。使用紧密融合的内皮单层的研究将有助于确定的动态结构，如肌动蛋白丰富lamellipodia和内皮屏障功能的横弧应力纤维的角色。此外，内皮细胞表达GFP -肌动蛋白或其他允许其他亚细胞结构的可视化的融合蛋白的活细胞成像将提供详细的时空信息需要了解的信令和结构机制的Determine屏障的完整性。

<table border="1"><tbody><tr><td colspan="4"> 1。林格5倍股 </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>氯化钠</td><td>电解二氧化锰</td><td> SX0420 - 3 </td><td> 35摹</td></tr><tr><td>氯化钾</td><td> JT贝克</td><td> 3040 </td><td> 1.75摹</td></tr><tr><td>氯化钙</td><td>西格玛</td><td> C - 3881 </td><td> 1.47摹</td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M - 9397 </td><td> 1.44摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2。 MOPS缓冲 </td></tr><tr><td> 化学 </td><td>公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td> MOPS </td><td>西格玛</td><td> M3183 </td><td> 125.6摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3。白蛋白的生理盐溶液（APSS） </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>林格股票（5X） </td><td> N / A </td><td> N / A </td><td> 200毫升</td></tr><tr><td> MOPS缓冲</td><td> N / A </td><td> N / A </td><td> 5毫升</td></tr><tr><td>磷酸钠</td><td>西格玛</td><td> S - 9638 </td><td> 0.168摹</td></tr><tr><td>丙酮酸钠&lt;/ TD&gt; <td>西格玛</td><td> P5280 </td><td> 0.22摹</td></tr><tr><td> EDTA钠盐</td><td>西格玛</td><td> ED2SS </td><td> 0.0074摹</td></tr><tr><td>血糖</td><td>西格玛</td><td> G7528 </td><td> 0.901摹</td></tr><tr><td>白蛋白，牛</td><td> USB </td><td> 10856 </td><td> 10克</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">调整pH至7.4，在37 °，然后放入高压灭菌瓶和储存在4℃的无菌过滤° C。 </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4。 0.9％的生理盐水 </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>氯化钠</td><td>电解二氧化锰</td><td> SX0420 - 3 </td><td> 9摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5。 1.5％的明胶溶液 </td></tr><tr><td>明胶来自猪皮肤</td><td>西格玛</td><td> G2500 </td><td> 15摹</td></tr><tr><td> 0.9％的生理盐水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">温暖的解决方案，以37 ° C至充分溶解的明胶。虽然仍然温暖，无菌过滤器进入蒸压瓶和存储在4 ° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

微血管内皮细胞中起着重要的作用，作为一种选择性渗透的阻隔液体和溶质。内皮细胞之间的粘附路口调节内皮细胞的通透性，许多研究已表明肌动蛋白细胞骨架，以确定交界的完整性 1-5的重要贡献。皮层肌动蛋白带被认为是维护稳定路口1，2，4，5的重要。相反，肌动蛋白应力纤维被认为是内皮细胞内产生的向心力紧张，削弱路口 2-5 。这一理论已被内皮细胞治疗与已知增加内皮细胞通透性的炎症介质，然后修复细胞和标签显微镜观察F - actin的研究基础上。然而，这些研究提供了肌动蛋白细胞骨架的作用非常有限的理解，因为固定细胞的图像只提供快照没有时间约肌动蛋白的结构5动态的信息。 活细胞成像允许的决定内皮屏障的完整性机制的研究肌动蛋白细胞骨架的动态性质纳入。这种方法的主要优点是各种炎症刺激内皮细胞中的肌动蛋白结构的影响，可在同一组的活细胞前和治疗后评估，消除潜在的偏见，观察固定标本时可能出现的的。的GFP -β-肌动蛋白的质粒转染人脐静脉内皮细胞（HUVEC）的盖玻片上生长汇合。汇合的人脐静脉内皮细胞肌动蛋白GFP的定时拍摄的图像前后此外，引起炎症介质的内皮屏障的完整性时间依赖性变化。这些研究使流体的肌动蛋白细胞骨架的变化的序列视觉观察到内皮贡献升屏障的破坏和恢复。 我们的研究结果一致显示，本地​​，肌动蛋白丰富lamellipodia内皮细胞的形成和营业额。也可以观察到肌动蛋白应力纤维的形成和运动。一个当地lamellipodia的形成和成交量的频率分析前和治疗后炎症刺激，可记录kymograph分析。这些研究提供了可以用于发现以前未知的分子机制的内皮屏障的完整性，维护重要内皮细胞的肌动蛋白细胞骨架的动态性质的重要信息。

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

显微成像可以表达GFP -肌动蛋白的内皮细胞骨架结构的动态变化特征。这种方法与使用固定标本的技术，在之前的相同细胞的肌动蛋白细胞骨架的时空变化提供了详细的评估，期间和之后，各种生理，药理，或炎症刺激。

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...