Role of Rac1 in Regulating the Integrity of Endothelial Adherens Junctions

The endothelium forms a semi-permeable barrier that is critical for maintaining tissue fluid homeostasis. Endothelial permeability if mainly regulated via Adherens Junctions (AJs) complex. The RhoGTPases Rac1 and RhoA play critical roles in regulating endothelial junctional permeability, however, the relationship between localized activity of RhoGTPases and the stability of Vascular Endothelial (VE)-cadherin adhesion, the main adhesive protein of AJs, remains unclear. Here using a photo-activatable probe to control spatiotemporal Rac1 activity at AJs, we addressed the relationship between Rac1 and dynamics of VE-cadherin in sub-confluent and confluent endothelium. We observed that in a sub-confluent monolayer Rac1 activation induced lamellipodia ruffling-a well-known effect of Rac1, and promoted gap sealing. Interestingly, Rac1 activation at mature junctions reduced the rate of VE-cadherin dissociation leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in RhoA-mediated and actomyosin-dependent tension across VE-cadherin adhesion sites and was independent of lamellipodia activity. Similar to Rac1 activation, inhibition of myosin II, downstream of RhoA, directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Both, in silico modeling and kinetic analysis of VE-cadherin mutant in cells, established the inverse relationship between stability of adhesive bonds and the time of VE-cadherin retention at AJs. Thus, Rac1 functioned by stabilizing VE-cadherin adhesion in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs resulting in the stabilizing of VE-cadherin adhesions.