Regulation of endothelial cell contacts during transendothelial migration of leukocytes

为了离开血流并进入组织，白细胞需要克服内皮细胞层的屏障。目前的研究方案解决了如何在分子水平上控制内皮细胞接触的开放和白细胞通过内皮细胞层的迁移的问题。三种完整的膜蛋白已被描述参与这一过程。抗VE-cadherin的抗体干扰内皮细胞层的完整性，促进白细胞在体外和体内的迁移，而针对PECAM-1和最近发现的连接黏附分子(JAM)的抗体则阻断白细胞的外渗，这在体外和体内再次得到证实。在这个项目的最后一个资助周期中，我们已经确定了JAM的两个细胞内结合伙伴，它们参与了细胞极性的建立。它们是含有蛋白AF-6(Afadin)和ASIP的PDZ结构域。AF-6是一种含有ras结合位点的肌动蛋白结合蛋白，而ASIP是线虫蛋白PAR-3的小鼠同源物，PAR-3是建立胚胎极性所必需的。最近证实，AF-6对于小鼠胚胎外胚层中紧密和粘连的连接的正确形成是必不可少的，从而导致胚胎死亡。我们计划分析这些蛋白在内皮细胞接触控制中的可能参与。此外，我们还将研究各种受体-磷酸酪氨酸磷酸酶(R-PTPs)与内皮细胞层通透性调节的相关性。其中一种R-PTP最近被鉴定为一种称为VE-PTP的内皮特异磷酸酶。除了对靶分子进行生化分析外，我们还将利用盖玻片中生长的内皮细胞建立的跨内皮细胞迁移试验，分析各种蛋白质可能参与白细胞外渗的可能性。结合计算机辅助的显微注射系统，我们将尝试通过过度表达候选分子或它们的突变来操纵通过注射细胞补丁的轮回。

In order to leave the blood stream and enter into tissues leukocytes need to overcome the barrier of the endothelial cell layer. The present research proposal addresses the question how the opening of endothelial cell contacts and the transmigration of leukocytes through the endothelial cell layer is controlled on the molecular level. Three integral membrane proteins have been described to be involved in this process. Antibodies against VE-cadherin interfere with the integrity of the endothelial cell layer and accelerate leukocyte migration in vitro as well as in vivo while antibodies against PECAM-1 and the recently identified Junctional Adhesion Molecule (JAM) block leukoyte extravasation, as was demonstrated again in vitro and in vivo. In the last funding periode of this project we have identified two intracellular binding partners of JAM, that are involved in the establishment of cell polarity. They are the PDZ domain containing proteins AF-6 (afadin) and ASIP. AF-6 is an actin binding protein containing a ras binding site and ASIP is the mouse homologue of the C. elegans protein PAR-3, that is essential for the establishment of embryo polarity. AF-6 was recently demonstrated to be essential for the proper formation of tight and adherens junctions in mouse embryonal ectoderm leading to embryonal lethality. We plan to analyse the possible involvement of these proteins in the control of endothelial cell contacts. In addition, we will study the relevance of various receptor-phospho-tyrosine phosphatases (R-PTPs) for the regulation of endothelial cell layer permeability. One of these R-PTPs was recently identified as an endothelial specific phosphatase called VE-PTP. Besides biochemical analysis of the target molecules we will analyse the possible involvement of the various proteins in leukocyte extravasation using a transendothelial migration asay established with endothelial cells grown in cover-slips. In combination with a computer-assisted microinjection system we will try to manipulate transmigration through a patch of injected cells by overexpressing candidate molecules or mutants of them.