Biochemical isolation and characterization of the lateral border recycling compartment from human endothelial cells and determination of its regulatory role for transendothelial migration of leukocytes
Final Report Abstract
PECAM-1 plays an important role in the transmigration of neutrophils, monocytes and natural killer cells, both in vivo and in vitro. Blocking PECAM-1 with anti-PECAM-1 antibodies or soluble PECAM-1-Fc chimera selectively inhibits transendothelial migration by 70-90%. In resting endothelial cells, PECAM-1 cycles constitutively between the cell surface and an internal PECAM-1-bearing compartment. During diapedesis, PECAM is directly targeted to the zone of the endothelial cell border that contacts the transmigrating monocytes, rather then recycling evenly along the lateral cell borders. EM analysis of the compartmental structure revealed that it consists out of several 50nm vesicular-like subunits, which are still connected to each other and to the surface plasma membrane. Goal of this project is the isolation and biochemical analysis of the PECAM-1 bearing compartmental vesicles by applying cell fractionation and protein biochemical procedures. The performance of a thorough biochemical analysis of compartmental membrane specific fragments and its protein components should reveal further insights into the compartmental role in the process of transendothelial migration. In the project so far a cell fractionation procedure has been established, which allowed a molecular differentiation between PECAM-1 proteins bound to the junctional surface plasma membrane and PECAM molecules stored inside the compartmental structures. PECAM-1 containing vesicles derived from the compartment have been enriched in specific interface-fractions obtained from sucrose and OPTI-Prep density gradients. In combination with an immunoprecipitation procedure, which allowed removing cell surface plasma membrane from the cell homogenate before or after density gradient centrifugation, PECAM-1 bearing vesicular fractions could be obtained. Western Blot analysis and membrane type specific enzymatic activity assays revealed that this PECAM-positive vesicular fractions showed a coenrichment for early and late endosomal protein markers but not for markers specific for Golgi, Endoplasmatic Reticulum and surface plasma membrane. 2D Gel experiments were performed on membrane material obtained from PECAM-1 positive vesicular fractions. In comparison to controls characteristic protein spots have been identified and analysed in a following MALDI-Tof fingerprint analysis. First experiments revealed that the intermediate protein vimentin is a highly enriched component in the vesicular fraction obtained from HUVEC and iHUVEC cell homogenates.
