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Organization and dynamics of subcellular functional domains as regulators of endothelial cell junctions

作为内皮细胞连接调节器的亚细胞功能域的组织和动态

基本信息

批准号：
34325042
负责人：
Professor Dr. Hans-Joachim Schnittler
金额：
--
依托单位：
Institut für Anatomie und Vaskuläre Biologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2006
资助国家：
德国
起止时间：
2005-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/34325042?language=en
关键词：
Organization dynamics subcellular functional domains

项目摘要

Dynamics of cell junctions are critical in many processes such as movement of individual cells within a monolayer and transmigration of leukocytes or tumor cells. Such phenomena require local regulation of cell junctions, mechanisms that still needs to be unraveled. Here we postulate the existence of subcellular functional domains at endothelial cell junctions. This principle is implemented in other cellular regulations such as proteasomes or focal adhesion sites. Subcellular functional domains of endothelial junction can be defined as small clusters of adhesion receptor (e.g. cadherins) that are associated with regulatory molecules (e.g. (VEGF-R; VEPTP) and thus might individually be regulated. This hypothesis is due to the following discoveries: By super resolution microscopy we identified small individual complexes of VE-cadherin, the backbone of adherens junctions, that appear pearl-necklace-like along the junctions. Those clusters fuse together e.g. after shear stress application and in turn increase the barrier function. Another type of subcellular functional domain is the junctions associated intermittent lamellipodia (JAIL) that typically form at gaps between individual VE-cadherin cluster, drive VE-cadherin dynamics and maintain barrier function. Furthermore Eps15 and caveolin-1 that are involved in controlling cell adhesion by beta-catenin sequestering also localize close to the individual VE-cadherin cluster and thus are suited to modulate VE-cadherin mediated cell adhesion clusters. In this project we aim to characterize the nano-architecture of endothelial junctions and investigate how the subcellular functional domains are dynamically regulated. In a first approach we will use structured illumination micrososcopy (SIM), total interference fluorescence microscopy (TIRFM) and high resolution microscopy such as direct stochastic optical reconstruction (dSTORM) and photoactivated localization microscopy (PALM) to characterize the adherens and tight junctions and its interaction with actin and vimentin-intermediate filaments in both cell culture and in vivo. In a second approach we aim to analyze the dynamics oft he VE-cadherin cluster and its dynamic interaction with actin and vimentin-intermediate filaments. Therefore, we willuse fluorescent-tagged fusion proteins that carry e.g. EGFP, mCherry, HALO, SNAP, DENDRA, or EOS. Fusion proteins will be expressed in endotheliaum by leniviral and adenoviral gene transfer and investigate by spinning disc confocal microscopy (SDCM) as well as by SIM, TIRF und FRAP. After time lapse recording cells will be fixed and further analysed with dSTORM, PALM, iPALM. The outcome of these studies might significantly help to generate novel concepts of how cell jucntions are regulated particularly at the subcellular level.

细胞连接的动力学在许多过程中是至关重要的，例如单个细胞在单层内的运动和白细胞或肿瘤细胞的迁移。这些现象需要细胞连接的局部调节，其机制仍然需要解开。在这里，我们假设在内皮细胞连接处存在亚细胞功能域。这一原理也适用于其他细胞调节，如蛋白酶体或粘着斑。内皮连接的亚细胞功能结构域可以定义为粘附受体（例如钙粘蛋白）的小簇，其与调节分子（例如（VEGF-R; VEPTP））相关，因此可以单独调节。这一假设是由于以下发现：通过超分辨率显微镜，我们确定了小的个体复合物VE-钙粘蛋白，粘附连接的骨干，出现珍珠项链般的沿着连接。这些簇例如在施加剪切应力之后融合在一起，并且继而增加屏障功能。另一种类型的亚细胞功能结构域是与间歇性板状伪足（JAIL）相关的连接，其通常在个体VE-钙粘蛋白簇之间的间隙处形成，驱动VE-钙粘蛋白动力学并维持屏障功能。此外，通过β-连环蛋白螯合参与控制细胞粘附的Eps 15和小窝蛋白-1也定位在接近单个VE-钙粘蛋白簇的位置，因此适合于调节VE-钙粘蛋白介导的细胞粘附簇。在这个项目中，我们的目标是表征内皮连接的纳米结构，并研究如何动态调节亚细胞功能域。在第一种方法中，我们将使用结构照明显微镜（SIM），全干涉荧光显微镜（TIRFM）和高分辨率显微镜，如直接随机光学重建（dSTORM）和光活化定位显微镜（PALM），以表征粘附和紧密连接及其与肌动蛋白和波形蛋白中间丝在细胞培养和体内的相互作用。在第二种方法中，我们的目标是分析VE-钙粘蛋白簇的动力学及其与肌动蛋白和波形蛋白中间丝的动态相互作用。因此，我们将使用携带例如EGFP、mCherry、HALO、SNAP、DENFLOW或EOS的荧光标记融合蛋白。融合蛋白将通过慢病毒和腺病毒基因转移在内皮中表达，并通过旋转圆盘共聚焦显微镜（SDCM）以及SIM、TIRF和FRAP进行研究。延时后，将记录细胞固定并用dSTORM、PALM、iPALM进一步分析。这些研究的结果可能会显着有助于产生新的概念，细胞jucntions是如何调节，特别是在亚细胞水平。