Cell junctions connect adjacent endothelial cells to each other and control paracellular permeability, changes in cell shape and cell migration. Endothelial cell junctions are divided into adherens junctions and tight junctions, as well as gap junctions, via which endothelial can directly communicate. Despite hundreds of existing reports, it is still unclear how junction regulation takes place. In particular, the large number of molecules involved complicates the analyses. The dynamics of cell junctions is until now a less investigated topic. This is largely due to problems in the genetic manipulation of endothelial cells. Our group is interested in uncovering the impact of junction dynamics in junction regulation under physiological and pathological conditions both in cell culture models and in vivo. Therefore, we have established a lentiviral-based gene transduction system that gives high transduction rates of more than 90% in endothelial cell cultures. This technique allows expression of fluorescently-tagged proteins in primary endothelial cells as well as endothelial cell lines, such as endothelioma knock-out cells. Using spinning disc microscopy we have investigated junction dynamics and regulation using live cell imaging under shear stress conditions contemporary with physiological and pathological stimulation. The analyses uncovered that cell junction proteins are highly dynamic even under resting conditions and undergo stimuli-specific changes.