Distinct Alterations in Dendritic Spine Morphology in the Absence of ß-Neurexins.

Dendritic spines are essential for synaptic function because they constitute the postsynaptic compartment of the neurons that receives the most excitatory input. The extracellularly shorter variant of the presynaptic cell adhesion molecules neurexins, ß-neurexin, has been implicated in various aspects of synaptic function, including neurotransmitter release. However, its role in developing or stabilizing dendritic spines as fundamental computational units of excitatory synapses has remained unclear. Here, we show through morphological analysis that the deletion of ß-neurexins in hippocampal neurons in vitro and in hippocampal tissue in vivo affects presynaptic dense-core vesicles, as hypothesized earlier, and, unexpectedly, alters the postsynaptic spine structure. Specifically, we observed that the absence of ß-neurexins led to an increase in filopodial-like protrusions in vitro and more mature mushroom-type spines in the CA1 region of adult knockout mice. In addition, the deletion of ß-neurexins caused alterations in the spine head dimension and an increase in spines with perforations of their postsynaptic density but no changes in the overall number of spines or synapses. Our results indicate that presynaptic ß-neurexins play a role across the synaptic cleft, possibly by aligning with postsynaptic binding partners and glutamate receptors via transsynaptic columns.

JAM-A interacts with α3ß1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells.

Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.

Autoregulatory "Multitasking" at Endothelial Cell Junctions by Junction-Associated Intermittent Lamellipodia Controls Barrier Properties.

Vascular endothelial cell (EC) junctions are key structures controlling tissue homeostasis in physiology. In the last three decades, excellent studies have addressed many aspects of this complex and highly dynamic regulation, including cell signaling, remodeling processes of the proteins of tight junctions, adherens junctions, and gap junctions, the cytoskeleton, and post-transcriptional modifications, transcriptional activation, and gene silencing. In this dynamic process, vascular endothelial cadherin (VE-cadherin) provides the core structure of EC junctions mediating the physical adhesion of cells as well as the control of barrier function and monolayer integrity via remodeling processes, regulation of protein expression and post-translational modifications. In recent years, research teams have documented locally restricted dynamics of EC junctions in which actin-driven protrusions in plasma membranes play a central role. In this regard, our research group showed that the dynamics of VE-cadherin is driven by small (1-5 µm) actin-mediated protrusions in plasma membranes that, due to this specific function, were named "junction-associated intermittent lamellipodia" (JAIL). JAIL form at overlapping, adjacent cells, and exactly at this site new VE-cadherin interactions occur, leading to new VE-cadherin adhesion sites, a process that restores weak or lost VE-cadherin adhesion. Mechanistically, JAIL formation occurs locally restricted (1-5 µm) and underlies autoregulation in which the local VE-cadherin concentration is an important parameter. A decrease in the local concentration of VE-cadherin stimulates JAIL formation, whereas an increase in the concentration of VE-cadherin blocks it. JAIL mediated VE-cadherin remodeling at the subjunctional level have been shown to be of crucial importance in angiogenesis, wound healing, and changes in permeability during inflammation. The concept of subjunctional regulation of EC junctions is strongly supported by permeability assays, which can be employed to quantify actin-driven subjunctional changes. In this brief review, we summarize and discuss the current knowledge and concepts of subjunctional regulation in the endothelium.

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

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-ß by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-ß levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-ß has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-ß turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.

MAGI1 Mediates eNOS Activation and NO Production in Endothelial Cells in Response to Fluid Shear Stress.

Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE-cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1-mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress-induced and PKA/AMPK dependent eNOS activation and NO production.

A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function.

Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcellular level even within one cell. Although it is assumed that this dynamic remodeling is associated with a local change in endothelial barrier function, a direct proof is missing mainly because of a lack of appropriate experimental techniques. Here, we describe a new assay to dynamically measure local endothelial barrier function with a lateral resolution of ∼15 µm and a temporal resolution of 1 min. In this setup, fluorescence-labeled molecules are added to the apical compartment of an endothelial monolayer, and the penetration of molecules from the apical to the basal compartment is recorded by total internal reflection fluorescence microscopy utilizing the generated evanescent field. With this technique, we found a remarkable heterogeneity in the local permeability for albumin within confluent endothelial cell layers. In regions with low permeability, stimulation with the proinflammatory agent histamine results in a transient increase in paracellular permeability. The effect showed a high variability along the contact of one individual cell, indicating a local regulation of endothelial barrier function. In regions with high basal permeability, histamine had no obvious effect. In contrast, the barrier-enhancing drug forskolin reduces the permeability for albumin and dextran uniformly along the cell junctions. Because this new approach can be readily combined with other live cell imaging techniques, it will contribute to a better understanding of the mechanisms underlying subcellular junctional reorganization during wound healing, inflammation, and angiogenesis.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

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

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 for-mation 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.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

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

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. Fluorescence-based 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.

The CellBorderTracker, a novel tool to quantitatively analyze spatiotemporal endothelial junction dynamics at the subcellular level.

Endothelial junctions are dynamic structures organized by multi-protein complexes that control monolayer integrity, homeostasis, inflammation, cell migration and angiogenesis. Newly developed methods for both the genetic manipulation of endothelium and microscopy permit time-lapse recordings of fluorescent proteins over long periods of time. Quantitative data analyses require automated methods. We developed a software package, the CellBorderTracker, allowing quantitative analysis of fluorescent-tagged cell junction protein dynamics in time-lapse sequences. The CellBorderTracker consists of the CellBorderExtractor that segments cells and identifies cell boundaries and mapping tools for data extraction. The tool is illustrated by analyzing fluorescent-tagged VE-cadherin the backbone of adherence junctions in endothelium. VE-cadherin displays high dynamics that is forced by junction-associated intermittent lamellipodia (JAIL) that are actin driven and WASP/ARP2/3 complex controlled. The manual segmentation and the automatic one agree to 90 %, a value that indicates high reliability. Based on segmentations, different maps were generated allowing more detailed data extraction. This includes the quantification of protein distribution pattern, the generation of regions of interest, junction displacements, cell shape changes, migration velocities and the visualization of junction dynamics over many hours. Furthermore, we demonstrate an advanced kymograph, the J-kymograph that steadily follows irregular cell junction dynamics in time-lapse sequences for individual junctions at the subcellular level. By using the CellBorderTracker, we demonstrate that VE-cadherin dynamics is quickly arrested upon thrombin stimulation, a phenomenon that was largely due to transient inhibition of JAIL and display a very heterogeneous subcellular and divers VE-cadherin dynamics during intercellular gap formation and resealing.

Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.

Experimental tools to monitor the dynamics of endothelial barrier function: a survey of in vitro approaches.

Endothelial cells line the inner surface of all blood vessels and constitute a selective barrier between blood and tissue. Permeation of solutes across the endothelial cell monolayer occurs either paracellularly through specialized endothelial cell-cell junctions or transcellularly via special transport mechanisms including transcytosis, via the formation of transcellular channels, or by cell membrane transport proteins. Several in vitro assays have been developed in the past few decades to analyze the molecular mechanisms of transendothelial permeability. Measurement of the electrical resistance of the cell monolayer has proven to be particularly suitable for analyzing paracellular barrier function with high-time resolution over long time periods. We review the various permeability assays and focus on the electrical impedance analysis of endothelial cell monolayers. We also address current progress in the development of techniques used to investigate endothelial permeability with high-lateral resolution and under mechanical loads.

Actin filament dynamics and endothelial cell junctions: the Ying and Yang between stabilization and motion.

The vascular endothelium is a cellular interface between the blood and the interstitial space of tissue, which controls the exchange of fluid, solutes and cells by both transcellular and paracellular means. To accomplish the demands on barrier function, the regulation of the endothelium requires quick and adaptive mechanisms. This is, among others, accomplished by actin dynamics that interdependently interact with both the VE-cadherin/catenin complex, the main components of the adherens type junctions in endothelium and the membrane cytoskeleton. Actin filaments in endothelium are components of super-structured protein assemblies that control a variety of dynamic processes such as endo- and exocytosis, shape change, cell-substrate along with cell-cell adhesion and cell motion. In endothelium, actin filaments are components of: (1) contractile actin bundles appearing as stress fibers and junction-associated circumferential actin filaments, (2) actin networks accompanied by endocytotic ruffles, lamellipodia at leading edges of migrating cells and junction-associated intermittent lamellipodia (JAIL) that dynamically maintain junction integrity, (3) cortical actin and (4) the membrane cytoskeleton. All these structures, most probably interact with cell junctions and cell-substrate adhesion sites. Due to the rapid growth in information, we aim to provide a bird's eye view focusing on actin filaments in endothelium and its functional relevance for entire cell and junction integrity, rather than discussing the detailed molecular mechanism for control of actin dynamics.

ARP2/3-mediated junction-associated lamellipodia control VE-cadherin-based cell junction dynamics and maintain monolayer integrity.

Maintenance and remodeling of endothelial cell junctions critically depend on the VE-cadherin/catenin complex and its interaction with the actin filament cytoskeleton. Here we demonstrate that local lack of vascular endothelial (VE)-cadherin at established cell junctions causes actin-driven and actin-related protein 2/3 complex (ARP2/3)-controlled lamellipodia to appear intermittently at those sites. Lamellipodia overlap the VE-cadherin-free adjacent plasma membranes and facilitate formation of new VE-cadherin adhesion sites, which quickly move into the junctions, driving VE-cadherin dynamics and remodeling. Inhibition of the ARP2/3 complex by expression of the N-WASP (V)CA domain or application of two ARP2/3 inhibitors, CK-548 and CK-666, blocks VE-cadherin dynamics and causes intercellular gaps. Furthermore, expression of carboxy-terminal-truncated VE-cadherin increases the number of ARP2/3-controlled lamellipodia, whereas overexpression of wild-type VE-cadherin largely blocks it and decreases cell motility. The data demonstrate a functional interrelationship between VE-cadherin-mediated cell adhesion and actin-driven, ARP2/3-controlled formation of new VE-cadherin adhesion sites via intermittently appearing lamellipodia at established cell junctions. This coordinated mechanism controls VE-cadherin dynamics and cell motility and maintains monolayer integrity, thus potentially being relevant in disease and angiogenesis.

Early Staphylococcus aureus-induced changes in endothelial barrier function are strain-specific and unrelated to bacterial translocation.

The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.

Imbalance of SMC1 and SMC3 cohesins causes specific and distinct effects.

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

Caveolin-1 opens endothelial cell junctions by targeting catenins.

AIMS: A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown. METHODS AND RESULTS: Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with ß- and γ-catenin. Consequently, the association of ß- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1. CONCLUSION: Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage.

Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm(2)) and arterial (12 dyn/cm(2)) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell-cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell-cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.

A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP1,2) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP1,2, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.