Dickinson, S.; (2006) Regulation of the actin cytoskeleton by ephrin-B signalling. Doctoral thesis , University of London.

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Abstract

Ephrin ligands and their Eph receptors play an essential role in angiogenesis during development. Both ephrins and Eph receptors are membrane-tethered proteins and their interaction at sites of cell-cell contact triggers bi-directional signalling, with signals transduced from the receptor (forward signalling) and ligand (reverse signalling). I have used two model systems to study ephrin-B2 signalling: Swiss 3T3 fibroblasts expressing exogenous ephrin-B2 and Human Umbilical Arterial Endothelial Cells (HUAECs) endogenously expressing ephrin-Bs. Stimulation of ephrin-B2 with soluble EphB receptors, has enabled the characterisation of the cellular responses, and signalling pathways, triggered by ephrin-B2 activation. I have shown that clustering of expressed ephrin-B2 in cultured fibroblasts induces a loss of cell-cell contact, dependent on the presence of serum factors and independent of actin-myosin contractility. The intracellular domain of ephrin-B2 is essential: tyrosine phosphorylation of the ligand via Src, and binding of the adaptor protein Grb4 are required for loss of cell-cell contact. Stimulation of endogenous ephrin-B2 in cultured endothelial cells results in dramatic cell retraction, and in a proportion of cells membrane blebbing. I have shown that the small GTPase Rho and activation of its downstream effector ROCK are essential for membrane retraction to occur, which is driven by an actin-myosin contraction event. In addition, I find that the c-Jun amino terminal kinase (JNK) is required for retraction, acting upstream of Rho/ROCK, and retraction occurs independently of Grb4. The cell contraction response to ephrin-B2 activation is rapid and transient with cells recovering to re-spread lamellipodia within minutes. Re-spreading is coupled to a loss of actin stress fibres and concomitant with down regulation of Rho and ROCK activity.

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