Probing Intercellular Interactions between Vascular Endothelial Cadherin Pairs at Single-molecule Resolution and in Living Cells

doi:10.1016/j.jmb.2006.02.021

Journal of Molecular Biology

Volume 358, Issue 3, 5 May 2006, Pages 665-674

https://doi.org/10.1016/j.jmb.2006.02.021 Get rights and content

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell–cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell–cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell–cell junctions.

Introduction

Adhesive junctions between neighboring endothelial cells are composed of transmembrane adhesive glycoproteins that are directly or indirectly linked to the cytoskeleton. These endothelial junction complexes are critical to the establishment and maintenance of the structural integrity of the vascular endothelium and control the permeability of the vasculature to small molecules. These endothelial junctions also serve as dynamic selective barriers to macromolecules and cells in the bloodstream.

Vascular endothelial cadherin (VE-cadherin) is the endothelial cell-specific cadherin, which localizes to adhesive intercellular endothelial junctions. VE-cadherin plays a key role in the remodeling, gating, and maturation of vascular vessels.1, 2 Cadherins are single-pass transmembrane glycoproteins, which associate as cis-dimers on the cell surface and combine to form structures that promote intercellular adhesions.³ Like other members of the cadherin family of cell-surface adhesion molecules, VE-cadherin molecules mediate calcium-dependent, homophilic adhesion between cells and function as plasma membrane attachment sites for the cytoskeleton through complex dynamic binding interactions of α-catenin and β-catenin to their cytoplasmic domain.4, 5 VE-cadherin is like classical type I cadherins, composed of five heptad extracellular domain repeats, a transmembrane domain, and a cytoplasmic domain that contains catenin and p120 binding sites. Nevertheless, VE-cadherin contains two extra introns on the sequence coding for the extracellular regions, which are not found in other members of the cadherin family.⁶ VE-cadherin is grouped within type II cadherins on the basis of its genomic structure.

Whether different members of the cadherin family show different binding capacity has been the subject of much debate. Differential binding capacity and adhesion specificity of cadherins are thought to mediate the formation of tissue boundaries and cell sorting during the development of solid tissues.7, 8 However, recent results suggest that cadherins are more promiscuous than previously suspected. Cadherins seem to be able to bind through heterophilic interactions.9, 10 A cell sorting assay and a flow chamber assay, where cultured cells expressing different types of surface cadherins were allowed to flow over (and potentially adhere to) recombinant cadherins immobilized on glass. Results from these measurements suggest that the extent of cell sorting observed in vitro correlates with neither binding capacity nor adhesion specificity.¹¹

Here, we characterize the binding kinetics and the micromechanical properties of the VE-cadherin bonds at the single-molecule level and in living human endothelial cells. The chosen system has the appeal of consistently probing VE-cadherin interactions between human umbilical vascular endothelial cells (HUVECs). We compare the single-molecule binding capacity of VE-cadherins to those of epithelial (E) cadherins and neuronal (N) cadherins, the two prototypical type I cadherins. Here, we use a sensitive nanoscale micromanipulation method that directly measures the adhesion force of individual bonds made of single VE-cadherin pairs expressed on the surface of living HUVECs. The single-molecule analysis presented here resolves pairwise molecular interactions from global cell–cell interactions, which can be measured by the flow chamber assay,¹¹ the dual pipette assay,12, 13 or cell aggregation assays.⁸ Our use of living cells rather than recombinant proteins14, 15, 16 ensures that the natural orientation of cadherins on the cell surface is preserved and it ensures that post-translational modifications of cell surface molecules, such as glycosylation, are also preserved.

We compare the kinetics and micromechanical properties of VE-cadherin/VE-cadherin bonds to those of the bonds made by prototypical classical type I cadherins, including E-cadherin and N-cadherin. These single-molecule force spectroscopy measurements reveal that VE-cadherins form bonds that are significantly stronger when subjected to low loading rates and have a significantly longer lifetime than those formed by E-cadherin pairs and N-cadherin pairs, properties that are particularly suitable for their barrier function in the endothelium.

Section snippets

Resolving VE-cadherin/VE-cadherin binding interactions at single-molecule resolution in live HUVECs

VE-cadherin/VE-cadherin pairwise binding interactions on adjoining living cells were analyzed at the single-molecule level using a non-imaging molecular force probe (MFP) (Figure 1), which is close in architecture to the atomic force microscope (AFM) used extensively for imaging in biology. Individual biotinylated HUVECs were placed with the help of a microneedle onto the surface of a streptavidin-coated cantilever. Subsequently, the HUVEC at the tip of the cantilever was gently placed in

Discussion

VE-cadherin is a member of the cadherin multigene family of cell-surface adhesion molecules, which mediate cell–cell adhesion in solid tissues. VE-cadherin is expressed specifically by endothelial cells and plays a major role in the remodeling, gating, and maturation of vascular vessels. Whether VE-cadherin molecules form intercellular bonds that have kinetics and micromechanical properties that are different from those of other classical cadherins is unknown. Here we use a live-cell force

Cell culture

HUVEC cells (ATCC, Manassas, VA) were cultured in Ham's F12K medium with 2 mM l-glutamine adjusted to contain 1.5 g/l of sodium bicarbonate and supplemented with 0.1 mg/ml of heparin, 0.05 mg/ml of endothelial cell growth supplement (ECGS), and 10% (v/v) fetal bovine serum (HUVEC's complete growth medium). Cell cultures were maintained at 37 °C in a humidified, 5% CO₂ environment. Immediately before the MFP experiment, the medium was changed to serum-free medium containing

Acknowledgements

This work was funded by grants from the National Science Foundation (CTS0210718), the National Aeronautics and Space Administration (NAG9-1563), and the National Institutes of Health (GM075305 and CA101135), and a Howard Hughes Medical Institute graduate training grant.

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Journal of Molecular Biology

Probing Intercellular Interactions between Vascular Endothelial Cadherin Pairs at Single-molecule Resolution and in Living Cells

Introduction

Section snippets

Resolving VE-cadherin/VE-cadherin binding interactions at single-molecule resolution in live HUVECs

Discussion

Cell culture

Acknowledgements

J. Biol. Chem.

Blood

Cell

J. Mol. Biol.

Cell

Dev. Biol.

J. Biol. Chem.

Biophys. J.

Curr. Biol.

Curr. Opin. Struct. Biol.

Biophys. J.

J. Biol. Chem.

Dev. Biol.

Curr. Opin. Cell Biol.

Cell

Neuron

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow

J. Cell Biol.

VE-cadherin: adhesion at arm's length

Am. J. Physiol. Cell Physiol.