Adherens junctions connect stress fibres between adjacent endothelial cells
© Millán et al; licensee BioMed Central Ltd. 2010
Received: 4 April 2009
Accepted: 2 February 2010
Published: 2 February 2010
Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis and homeostasis. Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. However, in the endothelium, cell-cell junctions are highly dynamic and morphologically heterogeneous.
We report that endothelial cell-cell junctions can attach to the ends of stress fibres instead of to cortical F-actin, forming structures that we name discontinuous adherens junctions (AJ). Discontinuous AJ are highly dynamic and are increased in response to tumour necrosis factor (TNF)-α, correlating with the appearance of stress fibres. We show that vascular endothelial (VE)-cadherin/β-catenin/α-catenin complexes in discontinuous AJ are linked to stress fibres. Moreover, discontinuous AJ connect stress fibres from adjacent cells independently of focal adhesions, of which there are very few in confluent endothelial cells, even in TNF-α-stimulated cells. RNAi-mediated knockdown of VE-cadherin, but not zonula occludens-1, reduces the linkage of stress fibres to cell-cell junctions, increases focal adhesions, and dramatically alters the distribution of these actin cables in confluent endothelial cells.
Our results indicate that stress fibres from neighbouring cells are physically connected through discontinuous AJ, and that stress fibres can be stabilized by AJ-associated multi-protein complexes distinct from focal adhesions.
Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis. A major role of the vascular endothelium is to control the movement of small solutes and leukocytes in and out of the bloodstream. Endothelial junctions consist of several different multi-protein complexes, whose relative abundance and roles in regulating permeability and leukocyte diapedesis depend on the endothelial cell type. Endothelial adherens junctions (AJ) and tight junctions (TJ) are the main regulators of paracellular permeability in the endothelium. Some junctional proteins unique to endothelial cells, including PECAM-1, ICAM-2 and S-endo I, also contribute to endothelial barrier function . In endothelial AJ, the transmembrane vascular endothelial (VE)-cadherin binds the cytoplasmic proteins β-catenin and p120-catenin. β-catenin also binds α-catenin, which could link the AJ complex to actin filaments . However, the established model of a direct link between cortical actin filaments (F-actin) and α-catenin in AJ in epithelial cells has been questioned by data demonstrating that the binding of α-catenin to β-catenin or F-actin is mutually exclusive, and suggesting that α-catenin stabilizes AJ by regulating actin polymerization instead of by linking F- actin to AJ [3, 4].
Endothelial cell-cell junctions are regulated by a variety of extracellular stimuli, which often act by inducing reorganization of the actin cytoskeleton. For example, Rho guanosine triphosphate (GTPases) and their targets, the rho serine/threonine kinases (ROCKs), stimulate actomyosin-based contractility, generating stress fibres and focal adhesion (FA) and thereby contribute to the rapid increase in endothelial permeability in response to thrombin and histamine [5–8]. Stress fibres generated in response to these stimuli also reorganize junctional complexes [9–11]. Pro-inflammatory stimuli such as tumour necrosis factor (TNF)-α also induce long-term changes to endothelial cell-cell junctions, actin stress fibre reorganization and an increase in permeability [12, 13].
Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. Here we describe the distinct properties of endothelial cell-cell junctions that localize at the ends of stress fibres that we name discontinuous AJ. These structures are distinct from focal adhesions, which are found at the ends of stress fibres in subconfluent endothelial cells. In response to TNF-α, association of stress fibres with discontinuous AJ, but not focal adhesions, is increased, suggesting that AJ may play a role stabilizing stress fibres in confluent endothelial cells.
Composition and dynamics of discontinuous AJ
Discontinuous AJ are associated with stress fibres at cell-cell borders
Discontinuous AJ are associated with the end of stress fibres independently of focal adhesions (FA)
Stress fibre tension mediates formation of discontinuous junctions
AJs are necessary for stress fibre attachment to cell-cell junctions
We show here that confluent HUVECs contain significantly less FA than subconfluent cells, even after stimulation with TNF-α which induces a large increase in stress fibres and overall F-actin content in confluent cells. Stress fibre formation is due to actomyosin contractility which requires them to be attached somewhere at both ends. Here we demonstrate that stress fibres are attached to VE-cadherin-mediated junctional complexes, although it is likely that some other junctional proteins are also linked to endothelial stress fibres, such as junctional adhesion molecule (JAM)-A or ZO-2-associated tight junctions. VE-cadherin engagement in confluent endothelial cells has been shown to reduce FA by modulating cell tension and spreading via RhoA . In accordance with this, VE-cadherin reduction by small interfering RNA (siRNA) significantly increased FA in confluent cells, although it did not alter the F-actin content. These results explain previous observations on the distribution of tyrosine phosphorylated proteins in endothelial cells which, in subconfluent cells, localized predominantly in a FA-like pattern whereas in confluent cells they were mostly at intercellular borders . The dynamic inter-conversion between linear and discontinuous AJ suggests that there could be a rapid tension-regulated switch of AJ proteins, which may be linked either to F-actin structures that protect the endothelial barrier, such as cortical F-actin , or to stress fibres. This link might be regulated, for example, by tension-induced unfolding of a linker protein in AJ, as has been postulated for p130Cas in FA .
Interestingly, cadherin complexes have been reported to associate with F-actin similar to stress fibres in transformed epithelial cells and endothelial cells undergoing remodelling (for example in response to wound healing or acute pro-inflammatory stimuli) [9–11, 26]. In epithelial cells, E-cadherin has also been shown not to associate with F-actin via α-catenin. It has been proposed that there is no direct association between AJ and the cortical F-actin , although it remains possible that another AJ protein can connect AJ to cortical F-actin. Here, based on the specific distribution of discontinuous AJ, together with an analysis of the dynamics of the AJ component p120-catenin and F-actin and the effect of VE-cadherin depletion on stress fibres, we propose that endothelial discontinuous AJ formed by complexes of VE-cadherin, α-catenin, β-catenin and p120-catenin, can be physically linked to actin stress fibres. It is possible that a tension-regulated AJ protein links AJ to stress fibres only under tension but not in resting conditions where linear endothelial AJ are more similar to epithelial AJ and co-localize with cortical F-actin . In agreement with this, stimuli that enhance endothelial barrier properties, such as cyclic adenosine monophosphate, increase the cortical F-actin belt but decrease stress fibres . Taken together, our results and those of others indicate that, both in endothelial and epithelial cells, the association of F-actins such as stress fibres with AJ is regulated and not constitutive. This regulated association would, for example, facilitate cell-cell junction remodelling during wound healing or in response to inflammatory stimuli.
Another role of endothelial stress fibres during inflammation is likely to be the regulation of leucocyte transmigration. Adhesion receptors involved in leucocyte adhesion, such as ICAM-1 or VCAM-1 and transmigration, align with stress fibres in response to engagement [28, 29]. ICAM-1 engagement increases RhoA activity and stress fibres  as well as regulating phosphorylation of VE-cadherin [30, 31]. ICAM-1 and VCAM-1 crosslinking alter the integrity of VE-cadherin cell contacts [31, 32]. In the light of these previous results, our data suggest that stress fibres are connectors from apically localized receptors to cell-cell junctions, which may contribute to leukocyte transmigration during inflammation. Stress fibres thus not only regulate endothelial permeability to small solutes, but may also help to withstand the mechanical stress generated by leukocyte transmigration under shear stress. We propose that the linkage of stress fibres between neighbouring cells via discontinuous AJ contributes to increase stress resistance and to regulate the whole endothelial monolayer response to inflammation.
The current model for cell-cell junctional organization is largely inspired by studies in epithelium. In epithelial cells AJ and TJ are separately organized and associated with cortical actin, although recently it has been proposed that AJ are not directly linked to cortical actin . The endothelium requires more dynamic and heterogeneous cell junctions in order to coordinate fast and local permeability increases to small molecules and cells from the bloodstream. Here we have shown that the ends of actin stress fibres, key actors in leukocyte transmigration and paracellular permeability regulation, are associated with cell-cell junctions. Cell-cell junctions can even connect stress fibres from neighbouring cells. Finally, we provide clear evidence that AJ are supra-molecular protein complexes distinct to FAs able to stabilize stress fibres in confluent endothelial cells. Our results clearly show a distinct organization of endothelial F-actin at confluence which is likely to be relevant during permeability changes and leukocyte transendothelial migration and for endothelial inflammatory migration and angiogenesis.
Mouse monoclonal anti-VE-cadherin blocking antibody was obtained from BD Pharmingen (CA, USA; Cat. 555661). Anti-VE-cadherin (Cat. 610252), anti-α-catenin, anti p120-catenin, anti-FAK, anti-phospho-Y397-FAK and anti-paxillin (610051) mouse monoclonal antibodies were obtained from BD Transduction Laboratories (NJ, USA). Anti-β-catenin (C-2206) rabbit polyclonal and anti-talin (T-3287) mouse monoclonal antibodies were obtained from Sigma (Melbourne, Australia). Anti-α-catenin rabbit polyclonal antibody was obtained from Santa Cruz (CA, USA). Anti-γ-catenin mouse monoclonal antibody, anti-ZO-1 and anti-JAM-A rabbit polyclonal antibodies were obtained from Zymed (CA, USA). Anti-pY118-paxillin phospho-specific antibody (44-722) was obtained from Biosource (Nivelles, Belgium).
Cell culture and transfection
HUVECs were obtained from Lonza (Wokingham, UK). They were cultured in Nunclon flasks pre-coated with 10 μg/ml human fibronectin in endothelial basal medium (EBM-2; (Lonza, MD, USA) supplemented with 2% fetal bovine serum (FBS), endothelial cell growth supplement EGM-2 (Lonza) (growth medium) in an atmosphere of 5% CO2/95% air. When experiments were performed in starving conditions, confluent HUVECs were starved in EBM-2 medium supplemented with 1% fetal calf serum (starvation medium) prior to stimulation with 10 ng ml-1 TNFα. No major differences in FA content were detected in cells stimulated in growth medium or starving medium.
HUVECs were transiently transfected with 1-5 μg plasmid DNA/106 cells with a Nucleofector kit (VPB-1002) (Amaxa Biosystems, Cologne, Germany) according to the manufacturer's instructions, and used for experiments 24 h to 72 h after transfection. For experiments with cells expressing different fluorescently-tagged proteins in the same monolayer, 3 × 106 cells were transfected with each plasmid, pooled together, plated on two glass-bottom 35-mm dishes (MatTek Corporation, MA, USA) or two glass coverslips previously coated with fibronectin for 15 h and analysed 24 to 48 h after transfection. Since nucleofection can induce significant cell death, once transfected cells were plated, untransfected cells were sometimes added in order to provide sufficient cells to form of a confluent monolayer.
For siRNA transfection, a protocol derived from a modification of our previous method  was used for delivery of siRNA with high efficiency into primary endothelial cells. HUVECs were plated at sub-confluence (105 cells on each well of a six-well dish) in EBM-2 medium with no antibiotics. The following day cells were transfected by mixing 4 μl of oligofectamine with siRNA to a final concentration of 100 nM. Twenty-four hours after transfection cells were trypsinized and plated at confluence onto different dishes in order to perform parallel assays such as immunofluorescence and western blotting. Assays were performed 72 h after transfection.
Plasmids and siRNAs
In order to construct the p120-DsRed plasmid, p-EGFP-120ctn- (generous gift from Keith Burridge) and dsRed vector from Clontech (CA, USA) were sequentially digested with AgeI and NotI enzymes and, then, the p120 vector without the GFP and the DsRed were ligated using the Ligase enzyme (New England Biolabs, Massachusetts, USA). β-Actin-GFP was a generous gift from Dr Beat Imhof. β-actin-Cherry was a generous gift from Ke Hu and Dr Ann Wheeler. The following siRNA oligonucleotides were obtained from the predesigned siGenome collection of Dharmacon (IL, USA). D-003641-01 (VE-cadherin), D-007746-01 (ZO-1). D-001210-01 (control (1)) or D-001810-01 (control (2)) non-targeting siRNA were used as controls in different experiments.
Confocal and time-lapse microscopy
For confocal microscopy, cells were fixed with 4% paraformaldehyde for 20 min, or at -20°C in 100% methanol for 5 min, for the detection of Talin. They were then blocked with tris-buffered saline (25 mM Tris pH 7.4, 150 mM NaCl) for 10 min, permeabilized for 5 min with phosphate buffered saline (PBS) containing 0.2% Triton X-100 at 4°C, blocked with PBS containing 1% bovine serum albumin and incubated at 37°C with primary then fluorophore-conjugated secondary antibodies or 1 μg/ml TRITC/FITC-labelled phalloidin. Specimens were mounted in DAKO fluorescent mounting medium (DAKO Corporation, CA, USA).
Confocal laser scanning microscopy was carried out with an LSM 510 (Zeiss, Welwyn Garden City, UK) mounted over an Axioplan microscope (Zeiss) using a ×40 1.3 NA oil immersion objective. In order to obtain Z stacks, three to six optical sections were taken over 4 μm. Intensity profiles were generated using the Zeiss LSM software.
Time-lapse microscopy was performed with a Nikon TE2000-E Eclipse Inverted microscope, in an environmental chamber at 37°C. 20 mM HEPES (pH 7.4) was added to the cells 2 h before the experiment. Images were taken with a cooled CCD camera Hamamatsu Orca-ER C4742-95. Camera and shutter (Lambda Instruments, VA, USA) were controlled by Andor Q software. Movies were processed with Metamorph software.
TNF-α-stimulated or unstimulated HUVECs were fixed for electron microscopy using 2% glutaraldehyde in 60 mM PIPES, 25 mM HEPES, pH 7.3, 3 mM MgCl2, 10 mM EGTA and 1% Triton TX-100, treated with 1% osmium tetroxide and dehydrated through a graded series of ethanol. The samples were then embedded in TAAB resin by conventional procedures, and 70 nm sections were cut using a Leica Ultracut E ultra microtome (Leica, Vienna, Austria). Sections were mounted onto 200 mesh copper grids and stained with lead citrate, before viewed on a H7600N transmission electron microscope (Leica). Digital images were captured using an AMT camera (Deben, Suffolk, UK).
Quantification of F-actin, FAs and discontinuous junctions
Confocal images from 8 to 30 cells per experiment from at least three different experiments were contrasted and analysed using LSM 510 software (Zeiss) in order to distinguish morphologically discontinuous junctions or FA-like paxillin staining at the basal planes. For F-actin quantification, images from cells stained for TRITC-labelled phalloidin were exported in formats compatible with ImageJ software. ImageJ was used to obtain the mean fluorescent intensities from different cellular regions of confluent cells, subconfluent cells or cells at the border of a wound induced in a confluent monolayer with a plastic tip 5 h before. Data were processed and statistical significance determined using Student's t-test (Microsoft Excel).
cyclic adenosine monophosphate
red fluoroescent protein
green fluorescent protein
human umbilical vein endothelial cells
phosphate buffered saline
small interfering RNA
tumour necrosis factor
focal adhesion kinase.
This work was supported by the Ludwig Institute for Cancer Research, Association for International Cancer Research (AICR) and the European Commission contract LSHG-CT-2003-502935 (MAIN). JM was supported by a Marie Curie fellowship (No. HPMF-CT-2000-01061) and a British Heart Foundation intermediate fellowship (No. FS/04/006), a grant from the Spanish Ministerio de Ciencia e Innovación (SAF2008-01936) and a Ramón y Cajal contract from the Spanish Government. NR is supported by a JAE predoctoral fellowship from CSIC (Spain). BM was supported by a FPI fellowship (SAF-2008-01936). We thank Alice Warley and Ken Brady (Centre for Ultrastructural Imaging, King's College London, UK) for technical assistance in the preparation of electron microscopy samples. We are grateful to Nicolas Reymond, Philippe Riou, Francisco Vega and other members of the Ridley laboratory, as well as to Juan Francisco Aranda and Miguel Alonso, for helpful discussions and technical support.
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