Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells

Abstract

In endothelial monolayers agonist-induced influx of Ca(2+) and activities of the actin cytoskeleton have been shown to be crucially involved in regulation of barrier properties. By laser tweezer application we demonstrated that the strength of adhesion of VE-cadherin-coated microspheres to the surface of cultured endothelial monolayers is significantly reduced by treatment with two well-established permeability-increasing compounds, cytochalasin D and the Ca(2+)-ionophore A23187, which shows that both compounds directly affect cadherin-mediated adhesion. Cytochalasin D and A23187 caused considerable decay of F-actin (30-60%). Stabilisation of F-actin by jasplakinolide completely blocked drug-induced weakening of bead adhesion showing that attenuation of cadherin-cadherin trans-interaction induced by cytochalasin D and A23187 depends largely on downregulation of F-actin. Single molecule fluorescence microscopy demonstrated that drug-induced weakening of adhesion is accompanied by an increase in lateral mobility of cadherins as well as by dispersal of cadherin-enriched plasmalemmal microdomains. However, the lifetime ( approximately 700 milliseconds, k(off) approximately 1.4 second(-1)) and apparent on-rate of cadherin trans-interaction (relative frequency of binding) remained unchanged in response to cytochalasin D and A23187 indicating that cadherin-mediated adhesion is not modulated by inside-out changes of the affinity but, rather, appears to be controlled by actin-dependent tethering and compartmentalization of cadherins.