Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium

Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent pr otein fusion construct (VEcadGFP) that mimics the native molecule, we visua lized alterations in endothelial junctional structure in real time during t ransmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant transmigration occurring exclusively at the cell bord ers (paracellularly). Surprisingly, transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4-6 mum in size were formed aft er a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contri buting to a junction. Gaps rapidly resealed within 5 min after leukocyte tr ansmigration. Migrating leukocytes appeared to push aside VEcadGFP in the p lane of the junction, and this displaced material subsequently diffused bac k to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time du ring transmigration.