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

Abstract

In endothelial monolayers agonist-induced influx of Ca2+ 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 Ca2+-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 (≈700 milliseconds, koff≈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.

Membrane and acto-myosin tension promote clustering of adhesion proteins

“…In contrast, in the case of vinculin, the cell can regulate the assembly of adhesive structures via a cytoplasmic protein complex. Such a regulation can also exist with cadherins as shown by experiments using beads covered with cadherins (20)(21)(22)(23). In this setup, cadherins are not just behaving as glue.…”

Cytoskeletal restraints regulate homotypic ALCAM-mediated adhesion through PKCα independently of Rho-like GTPases

“…CytD, but not ALCAM-mediated adhesion, causes activation of RhoA, Rac1 and Cdc42 Disruption of the actin cytoskeleton by CytD treatment results in increased lateral mobility and cluster formation of integrins (Elemer and Edgington, 1994;Lub et al, 1995), cadherins (Baumgartner et al, 2003), and other cell adhesion molecules, such as Ep-CAM (Balzar et al, 1998), leading to increased adhesion. In addition to disrupting the actin cytoskeleton, Ren and co-workers (Ren et al, 1999) showed that in quiescent Swiss 3T3 fibroblasts CytD treatment activates RhoA.…”

Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions

“…At the later stages of assembly, a submembranous sheet of Dia1-nucleated actin filaments could increase adhesion by reducing the lateral mobility of cadherin receptors in the plane of the membrane; this mechanism does not require a direct link between cadherin receptors and the actin filaments (Baumgartner et al, 2003;Iino et al, 2001;Sako et al, 1998). Another interesting possibility is the actin-polymerizationdependent control of turnover of adhesion receptors.…”