Organization and dynamics of subcellular functional domains as regulators of endothelial cell junctions

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Dynamics of cell junctions are critical in many processes such as movement of individual cells within a monolayer and transmigration of leukocytes or tumor cells. Such phenomena require local regulation of cell junctions, mechanisms that still needs to be unraveled. Here we postulate the existence of subcellular functional domains at endothelial cell junctions. This principle is implemented in other cellular regulations such as proteasomes or focal adhesion sites. Subcellular functional domains of endothelial junction can be defined as small clusters of adhesion receptor (e.g. cadherins) that are associated with regulatory molecules (e.g. (VEGF-R; VEPTP) and thus might individually be regulated. This hypothesis is due to the following discoveries: By super resolution microscopy we identified small individual complexes of VE-cadherin, the backbone of adherens junctions, that appear pearl-necklace-like along the junctions. Those clusters fuse together e.g. after shear stress application and in turn increase the barrier function. Another type of subcellular functional domain is the junctions associated intermittent lamellipodia (JAIL) that typically form at gaps between individual VE-cadherin cluster, drive VE-cadherin dynamics and maintain barrier function. Furthermore Eps15 and caveolin-1 that are involved in controlling cell adhesion by beta-catenin sequestering also localize close to the individual VE-cadherin cluster and thus are suited to modulate VE-cadherin mediated cell adhesion clusters. In this project we aim to characterize the nano-architecture of endothelial junctions and investigate how the subcellular functional domains are dynamically regulated. In a first approach we will use structured illumination micrososcopy (SIM), total interference fluorescence microscopy (TIRFM) and high resolution microscopy such as direct stochastic optical reconstruction (dSTORM) and photoactivated localization microscopy (PALM) to characterize the adherens and tight junctions and its interaction with actin and vimentin-intermediate filaments in both cell culture and in vivo. In a second approach we aim to analyze the dynamics oft he VE-cadherin cluster and its dynamic interaction with actin and vimentin-intermediate filaments. Therefore, we willuse fluorescent-tagged fusion proteins that carry e.g. EGFP, mCherry, HALO, SNAP, DENDRA, or EOS. Fusion proteins will be expressed in endotheliaum by leniviral and adenoviral gene transfer and investigate by spinning disc confocal microscopy (SDCM) as well as by SIM, TIRF und FRAP. After time lapse recording cells will be fixed and further analysed with dSTORM, PALM, iPALM. The outcome of these studies might significantly help to generate novel concepts of how cell jucntions are regulated particularly at the subcellular level.