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移行。这种现象需要对细胞连接进行局部调控，而这一机制仍需解开。在此，我们假设内皮细胞连接处存在亚细胞功能区。这一原理在其他细胞调节中也有应用，如蛋白酶体或粘着斑点。内皮细胞连接的亚细胞功能区可以被定义为黏附受体(如钙粘附素)的小簇，它们与调节分子(如血管内皮生长因子受体；VEPTP)相关，因此可能单独受到调控。这一假说归因于以下发现：通过超分辨率显微镜，我们发现了VE-钙粘蛋白的小的单个复合体，它是粘附素连接的主干，沿着连接呈现珍珠项链状。例如，在施加剪应力之后，这些团簇融合在一起，进而增加了屏障功能。另一种类型的亚细胞功能结构域是与间歇性片状脂蛋白(JAR)相关的连接，通常形成于单个VE-钙粘蛋白簇之间的缝隙处，驱动VE-钙粘蛋白动态变化并维持屏障功能。此外，参与控制细胞黏附的Eps15和小窝蛋白-1也位于单个VE-钙粘蛋白簇附近，因此适合于调节VE-钙粘蛋白介导的细胞黏附簇。在这个项目中，我们的目标是描述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)和光活化定位显微镜(Palm))来表征细胞培养和体内的贴壁和紧密连接及其与肌动蛋白和波形蛋白中间丝的相互作用。在第二种方法中，我们旨在分析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，我们将使用带有荧光标记的融合蛋白，例如，EGFP、mCherry、Halo、SNAP、Dendra或EOS。融合蛋白将通过腺病毒和腺病毒基因转移在内皮细胞中表达，并通过旋转圆盘共聚焦显微镜(SDCM)以及SIM、TIRF和FRAP进行研究。经过时间推移后，记录细胞将被固定，并进一步用dSTORM，Palm，iPALM进行分析。这些研究的结果可能大大有助于产生关于细胞连接如何调节的新概念，特别是在亚细胞水平上。

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.