The role of Rap1 in cell-cell junction formation

Abstract

Both epithelial and endothelial cells form cell-cell junctions at the cell-cell contacts to maintain tissue integrity. Proper regulation of cell-cell junctions is required for the organisation of the tissue and to prevent leakage of blood vessels. In endothelial cells, the cell-cell junctions are highly regulated to ensure the passage of leukocytes, large molecules and fluids over the endothelial barrier. Disruption of the endothelial barrier is involved in various pathological conditions, such as edema and inflammation. The small GTPase Rap1 is involved in the formation of cell-cell junctions in both epithelial and endothelial cells. Like most GTPases, Rap1 is activated by gunanine-nucleotide exchange factors (GEFs) and inactivated by GTPase activating proteins (GAPs). This thesis addresses the role of the Rap1 signalling network in the formation and regulation of cell-cell junctions. First, the role of two activators of Rap1 is addressed. The Rap1-GEF PDZ-GEF2 is required for the formation and maturation of cell-cell junctions both in epithelial and endothelial cells. Depletion of cells from PDZ-GEF2 results in zipper-like structures. The maturation of cell-cell junctions is dependent on Rap1a and not Rap1b, as Rap1a siRNA also results in zipper-like structures. Activation of Rap1 rescues the phenotype of PDZ-GEF2 depleted cells. Furthermore we could show that the Rap1-GEF exchange protein activated by cAMP (EPAC) is expressed in endothelial cells. Activation of Epac1 results in enhancement of the endothelial cell-cell junctions and the endothelial barrier. Besides the enhancement of endothelial cell-cell junctions, activation of Epac1 also results in the formation of cortical actin, independent of cell-cell junctions. In the second part of this thesis, we investigated the downstream signalling pathways involved in Rap1-mediated cell-cell junction formation. The Rap1 effector molecule AF-6 binds to Rap1, localises to cell-cell contacts and binds to various junctional proteins. However, knock-down of AF-6 has no effect on Epac1-induced endothelial cell-cell junction regulation. Interestingly, depletion of AF-6 results in reorganisation of the actin cytoskeleton, independently of cell-cell junctions. The endothelial barrier, measured by trans-endothelial electrical resistance is also impaired. Activation of Epac1 rescues the barrier defect caused by AF-6 depletion. To identify modifiers of the Rap1-mediated barrier regulation in endothelial cells, we used an siRNA based approach using trans-endothelial electrical resistance. We identified a few potential regulators of Epac1-induced barrier function. We investigated one of these regulators, Lamellipodin, in more detail. Lamellipodin is required for Epac1-induced endothelial resistance. Lamellipodin binds to the actin regulator VASP and activation of Epac1 results in translocation of VASP to the cell-cell contacts. Finally, we show that VE-cadherin is not required for Epac1-induced endothelial resistance, whereas an intact actin cytoskeleton is required. In addition to the novel results presented in this thesis, we discuss the current role of Rap1 in the regulation of junctions.