Quantitative dynamics of VE-cadherin at endothelial cell junctions at a glance: basic requirements and current concepts

Abstract

Intercellular junctions of the vascular endothelium are dynamic structures that display a high degree of plasticity, which is required to contribute to their regulation of many physiological and pathological processes including monolayer integrity, barrier function, wound healing and angiogenesis. Vascular endothelial cadherin (VE-cadherin) is connected via catenins to the actin cytoskeleton, both of which are key structures in endothelial junction regulation, and thus are the focus of much investigation. Fluorescencebased live cell imaging is the method of choice to study dynamic remodeling in living cells. Although these methods have been successfully applied to many cell types, investigations of endothelial junction dynamics were for a long time limited as they are largely resistant to transfection using many classical protocols. Application of virus-based gene transduction techniques, together with advanced microscopy, now allows both sufficient expression of fluorescence tagged junction-localized proteins in the endothelium and time-lapse recording over long periods. Using highly spatiotemporally resolved fluorescence microscopy it turned out that endothelial junctions display extensive junction heterogeneity at the subcellular level; a fact that largely limits automated quantification by available software. Recent work describes open software tools to quantitatively analyze large amounts of fluorescence-based image data in either single or confluent epithelial and endothelial cells. Based on quantitative VE-cadherin and actin dynamics novel key players, mechanisms and concepts have been suggested that control endothelial junction dynamics. Here we aim to summarize the recent developments in the field.

Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

“…The local permeability studies showed that permeated regions were most affected by the number of punctate and no junction regions of coverage, which had a positive correlation with PR area. This result supports the generally accepted idea that continuous junctions are indicative of a more mature and stable barrier [60][61][62]. This study also implicates punctate (versus perpendicular) junctions in the regulation of monolayer permeability, though future investigation is needed to unveil the differences between these two discontinuous junction types.…”

Putting VE-cadherin into JAIL for junction remodeling

“…The mechanisms underlying cell junction remodeling involve actin-and myosinmediated contractility, tyrosine phosphorylation of VE-cadherin, p120 ctn -mediated endocytosis and the formation of actin-driven plasma membrane protrusions (Doggett and Breslin, 2011; Hoelzle and Svitkina, 2012;Martinelli et al, 2013;Adderley et al, 2015). In addition, live-cell imaging is being increasingly used (Day and Davidson, 2009;Frigault et al, 2009;Cranfill et al, 2016;Seebach et al, 2016;Hofer et al, 2018), as it allows the direct observations of junction remodeling with high spatial and temporal resolution, thus providing valuable hints regarding the underlying mechanisms. In this context, our laboratory has focused on the interdependency between actin-driven membrane protrusions and VE-cadherin-mediated adhesion.…”

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging

“…The first problem relates to genetic manipulation of the endothelium, as these cells are hard to transfect by classical protocols. The use of viral vectors such as adenovirus and lentivirus vectors, however, has significantly increased the transfection rate and has been proven for sufficient application [for review, compare 11, 12]. Furthermore, if the dynamics of a protein interaction with different proteins is to be carried out, then in many cases different tags have to be cloned to the same protein.…”