Marston, Daniel John; (2004) Mechanisms for EphB4 regulated contact repulsion. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Eph receptor tyrosine kinases and their ligands, the ephrins, are essential regulators of cell migration in many developmental processes. They are distinct from most guidance cues in that both the Eph receptors and ephrins are membrane-attached and thus they interact with one another only at sites of cell-cell contact. The particular family member I have studied is EphB4 which, along with its ligand, the transmembrane protein ephrin-B2, plays a specific role in mammalian angiogenesis. Using cultured fibroblast and endothelial cells I have explored how EphB4 regulates the actin cytoskeleton via the Rho family of GTPases. I have demonstrated that activation of EphB4 with either soluble ephrin-B2 ectodomains, or ephrin-B2 expressed on neighbouring cells, stimulates Cdc42 and Rac regulated protrusive actin structures, filopodia and lamellipodia. Activation of EphB4 with soluble ephrin-B2 ectodomains also causes a loss of cell-matrix adhesion and cell retraction that is mediated by Rho signalling. The formation of the protrusive actin structures is inhibited by pre-clustering the ephrin-B2-Fc. Activation of EphB4 by ephrin-B2 expressed on an adjacent cell causes the receptor and ligand expressing cells to separate. The separation is concomitant with endocytosis of activated Eph receptors and their bound, full-length ephrinB ligands. Both the internalisation of the receptor-ligand complexes and the subsequent cell retraction events require Cdc42 and Rac-dependent actin polymerization within the receptor-expressing cells. Similar results are seen in cultured endothelial cells expressing endogenous EphB4, where expression of ephrin-B2 in isolated cells triggers Rac-dependent cell retraction and internalisation of activated Eph receptors into late endosomal compartments. My work suggests a novel mechanism for EphB/ephrinB mediated cell repulsion, in which the contact between cells is destabilised by the localised phagocytosis of the ephrin-expressing cell plasma membrane by the Eph receptor-expressing cell leading to cell-cell separation.

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