Actin tethering in endothelial junctions

In rat lung microvascular endothelial cells (RLMEC), enhancement of cortical actin (F‐actin) increases E‐cadherin at intercellular junctions, thereby enhancing endothelial barrier properties (Quadri, 2003). The Y12F mutation on the actin‐binding protein, α‐actinin (MUT‐α‐actinin) abrogates F‐actin formation. To determine actin‐cadherin interactions, we established confluent RLMEC monolayers expressing GFP‐E‐cadherin. Confocal microscopy of EC junctions revealed regions of high (HD) and low (LD) E‐cadherin density. To determine E‐cadherin mobility, we photobleached GFP fluorescence to 60% of initial, then determined fluorescence recovery (FRAP) at 5 min. At HD versus LD sites, FRAP was respectively, 28±3% versus 50±3% of the pre‐bleached fluorescence (P<0.05), indicating that E‐cadherin mobility was considerably lower at HD than LD sites. In monolayer expressing MUT‐α‐actinin, E‐cadherin mobility increased to 50±4% at HD sites (P<0.05), while remaining unchanged at LD sites, indicating that E‐cadherin‐F‐actin linkages were formed at HD sites alone. Our findings indicate that in lung endothelial cells, sites of high E‐cadherin density are focal locations of actin‐cadherin tether formation. The tether function is mediated by α‐actinin, which is therefore, potentially a critical regulator of endothelial barrier properties (Support: HL36024).