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

细胞连接的动力学在许多过程中是至关重要的，如单个细胞在单层内的运动和白细胞或肿瘤细胞的迁移。这种现象需要对细胞连接进行局部调控，而这一机制仍有待研究。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控，如蛋白酶体或局灶黏附位点。内皮连接的亚细胞功能域可以定义为粘附受体（如钙粘蛋白）的小簇，它们与调节分子（如VEGF-R、VEPTP）相关，因此可能单独受到调节。这一假设是由于以下发现：通过超分辨率显微镜，我们发现了小的单个ve -钙粘蛋白复合物，粘附连接的骨干，沿着连接呈珍珠项链状。这些团簇在剪切应力作用后融合在一起，从而增加了屏障功能。另一种类型的亚细胞功能域是连接相关的间歇性板足（JAIL），它通常形成于单个ve -钙粘蛋白簇之间的间隙，驱动ve -钙粘蛋白动力学并维持屏障功能。此外，参与通过β -catenin隔离控制细胞粘附的Eps15和caveolin-1也定位于靠近单个VE-cadherin簇的位置，因此适合调节VE-cadherin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，并研究亚细胞功能域是如何动态调节的。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）和光激活定位显微镜（PALM）来表征细胞培养和体内的粘附物和紧密连接及其与肌动蛋白和vimentin中间细丝的相互作用。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钙粘蛋白中间丝的动态相互作用。因此，我们将使用荧光标记的融合蛋白，如EGFP、mCherry、HALO、SNAP、DENDRA或EOS。融合蛋白将通过轻病毒和腺病毒基因转移在内皮细胞中表达，并通过旋转盘共聚焦显微镜（SDCM）以及SIM， TIRF和FRAP进行研究。延时后的记录单元将被固定并使用dSTORM， PALM， iPALM进一步分析。这些研究的结果可能有助于产生新的概念，特别是在亚细胞水平上如何调节细胞连接。

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.