Regulation of endothelial cell contacts during transendothelial migration of leukocytes

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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.