Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVβ3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and β3-integrin. Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.

中文翻译：

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αII-Spectrin 的缺乏会影响内皮细胞-基质的接触和迁移，导致体外血管生成受损。

细胞骨架成分的精确协调和细胞粘附和迁移的动态控制是关键细胞过程（如分化和形态发生）所必需的。我们研究了 αII-spectrin（一种普遍存在的膜骨架支架元件）在粘附和血管生成机制中的潜在参与。细胞模型是原代人脐静脉内皮细胞（HUVECs）和人真皮微血管内皮细胞系（HMEC-1）。在 siRNA 和 shRNA 介导的 αII-Spectrin 敲低后，我们使用蛋白质印迹评估了其表达及其伴侣和粘附蛋白的表达。使用免疫荧光和视频显微镜检查对照和血影蛋白耗尽细胞的表型。使用基于厚凝胶基质的方法和配备恒温室和 Nikon Biostation System 相机的显微镜评估毛细管形成。αII-spectrin 的敲除导致：改变细胞形状；存在外周肌动蛋白斑块的肌动蛋白细胞骨架组织；并减少应力纤维的形成。Spectrin缺乏会影响细胞对层粘连蛋白和纤连蛋白的粘附以及细胞运动。这包括改变粘附分子（如 αVβ3-和 α5-整合素）的定位，以及粘附结构（如焦点）的组织。正如血管生成模型所证明的，αII-Spectrin 的缺乏也会影响体外毛细管形成的复杂机制。实时成像显示，毛细管组件的损伤主要与细胞投影长度和稳定性的显着降低有关。αII-Spectrin 耗竭也与参与毛细管形成和组装的三种蛋白质的表达显着降低有关：VE-钙粘蛋白、MCAM 和 β3-整合素。我们的数据证实了 αII-spectrin 在控制细胞粘附和扩散中的作用。此外，我们的研究结果进一步支持 αII-Spectrin 通过控制粘附分子（如整合素）参与体外毛细管形成。这表明αII-血影蛋白在血管生成中的新功能。αII-Spectrin 耗竭也与参与毛细管形成和组装的三种蛋白质的表达显着降低有关：VE-钙粘蛋白、MCAM 和 β3-整合素。我们的数据证实了 αII-spectrin 在控制细胞粘附和扩散中的作用。此外，我们的研究结果进一步支持 αII-Spectrin 通过控制粘附分子（如整合素）参与体外毛细管形成。这表明αII-血影蛋白在血管生成中的新功能。αII-Spectrin 耗竭也与参与毛细管形成和组装的三种蛋白质的表达显着降低有关：VE-钙粘蛋白、MCAM 和 β3-整合素。我们的数据证实了 αII-spectrin 在控制细胞粘附和扩散中的作用。此外，我们的研究结果进一步支持 αII-Spectrin 通过控制粘附分子（如整合素）参与体外毛细管形成。这表明αII-血影蛋白在血管生成中的新功能。