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The Eph family of receptor tyrosine kinases and their cognate ligands, the Ephrins, form a coordinated program of cell contact-mediated migration control, polarity establishment, and tissue architecture development. Specifically, the ligand Ephrin-A1 has been shown to regulate cell morphology and motility through the activation of EphA receptors, which signal to the PI3K pathway to induce cell retraction. MEFs with PI3K subunit p85β knockout (p85β−/−) exhibited markedly reduced cell retraction following Ephrin-A1 stimulation. Ephrin-A1 also serves as an inhibitory substrate for cell spreading and migration. Moreover, Ephrin-A1 treatment results in the dephosphorylation of paxillin and induces the reorganization of phospho-paxillin-containing focal adhesions. The Ephrin-A1 regulated paxillin dephosphorylation is phosphatase dependent, but p85β independent. The present study serves to demonstrate a novel molecular signaling pathways that regulate Ephrin-A1-regulated cell retraction and interaction to the substrate.

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12195-009-0082-6) contains supplementary material, which is available to authorized users.