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

Abstract

Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for­mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.

EPLIN-α and -β Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin

“…There is EPLIN-a, which consists of 600 amino acids and a larger EPLIN-b isoform with 759 amino acids (Chen et al, 2000). Previous work on EPLIN in ECs demonstrated that both EPLIN isoforms localize as components of the JAAF, while EPLIN-a was additionally found at JAIL (Hofer et al, 2018), which is in agreement with its impact on angiogenesis (Chervin-Pé tinot et al, Sanders et al, 2010).…”

“…Cell migration and cell junction dynamics directly relate to formation of Arp2/3 complex-dependent plasma membrane protrusions including cLP and JAIL, respectively (for review, see Cao and Schnittler, 2019;Krause and Gautreau, 2014). Since EPLIN interferes with Arp2/3 complex function (Maul et al, 2003) and that EPLIN-a localizes at JAIL and cLP (Hofer et al, 2018), we investigated whether EPLIN isoform expression correlates with cell density in HUVEC cultures. Indeed, subconfluent EC cultures (3À4 3 10 4 cells/cm 2 ) expressed about 2 times more EPLIN-a than confluent cultures (0.9À1.2 3 10 5 cells/ cm 2 ), while EPLIN-b expression remained unchanged (Figure 1C).…”

“…Localization and dynamics of EPLIN isoforms were evaluated using EPLIN-a-mCherry, EPLIN-a-EGFP, EPLIN-b-mCherry, and EPLIN-b-EGFP (Hofer et al, 2018), which were expressed in HUVEC cultures and subsequently characterized by immunoprecipitations (IPs), by western blot analysis (Figures S2A-S2D), and by immune labeling (Figure S2E). Previous studies showed an association of EPLIN-a with the VE-cadherin/catenin complex (Chervin-Pé tinot et al, 2012), whereas both isoforms were localized at EC junctions in confluent HUVEC cultures (Hofer et al, 2018). In addition, using GFP-Trap_A assay, we demonstrate that both EPLIN-a-EGFP and EPLIN-b-EGFP co-precipitate a fraction of the VE-cadherin/catenin complex (Figure 1E), which consequently leads to a low but clear visible VE-cadherin signal.…”

“…Fluid shear stress causes a quick (minutes) and transient increase in EC barrier function, which accompany increased protrusion dynamics at the junctions in a Racdependent manner (DePaola et al, 2001;Hofer et al, 2018;Seebach et al, 2007). Therefore, we investigated this effect in relation to EPLIN localization and dynamics.…”

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.…”