Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin
Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin
复制标题
DOI：
10.3791/3187
复制
发表时间：
2011-11-01
影响因子：
1.2
通讯作者：
Breslin, Jerome W.
中科院分区：
文献类型：
--
作者：
Doggett, Travis M.;Breslin, Jerome W.
微血管内皮作为液体和溶质的选择性渗透屏障发挥着重要作用。内皮细胞之间的粘附连接调节内皮的通透性，许多研究表明肌动蛋白细胞骨架对确定连接完整性有重要贡献(1-5)。皮质肌动蛋白带被认为对于维持稳定连接很重要1,2,4,5。相反，肌动蛋白应力纤维被认为会在内皮细胞内产生向心张力，从而削弱连接(2-5)。该理论大部分基于以下研究：用已知可增加内皮通透性的炎症介质处理内皮细胞，然后固定细胞并标记 F-肌动蛋白以进行显微镜观察。然而，这些研究对肌动蛋白细胞骨架的作用的了解非常有限，因为固定细胞的图像仅提供及时的快照，没有有关肌动蛋白结构动态的信息(5)。活细胞成像可以将肌动蛋白细胞骨架的动态性质纳入决定内皮屏障完整性的机制的研究中。这种方法的一个主要优点是，可以在治疗前后在同一组活细胞中评估各种炎症刺激对内皮细胞肌动蛋白结构的影响，消除观察固定标本时可能出现的潜在偏差。用 GFP-β-肌动蛋白质粒转染人脐静脉内皮细胞 (HUVEC)，并在玻璃盖玻片上生长至汇合。在添加炎症介质之前和之后捕获汇合的 HUVEC 中 GFP-肌动蛋白的延时图像，这些炎症介质会引起内皮屏障完整性的时间依赖性变化。这些研究使得能够目视观察肌动蛋白细胞骨架变化的流体序列，这些变化有助于内皮屏障的破坏和恢复。我们的结果一致显示内皮细胞中局部富含肌动蛋白的板状伪足的形成和更新。还可以观察肌动蛋白应力纤维的形成和运动。可以通过 kymograph 分析记录在用炎症刺激治疗之前和之后局部片状伪足的形成和更新频率的分析。这些研究提供了有关内皮细胞中肌动蛋白细胞骨架动态性质的重要信息，可用于发现先前未识别的对于维持内皮屏障完整性很重要的分子机制。
The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity(1-5). A cortical actin belt is thought to be important for the maintenance of stable junctions1,2,4,5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions(2-5). Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures(5). Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-beta-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.