Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Isobel D. Lawrenson¹, Sabine H. Kleikamp¹, Peter Lock¹, Simone M. Schoenwaelder², Michelle Down³, Andrew W. Boyd³, Paul F. Alewood⁴ and Martin Lackmann¹

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Movie 1

Ephrin-A5 induced rounding and de-adhesion of AO9 melanoma cells. AO9 melanoma cells, plated to semi-confluency onto fibronectin-coated microscopic glass coverslips, were serum-starved and then stimulated by addition of clustered ephrin-A5 Fc to 10 nM final concentration. Microscopic images of a representative section of a slide were taken every minute during a time course 10 minutes before and 50 minutes after ephrin addition (indicated by a blacked-out frame at the beginning of the video) and processed using the Confocal Assistant Software (ver. 2.55, Bio-Rad) (see Fig. 2).

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Clustered ephrin-A5 triggers rapid cell rounding and dynamic membrane blebbing of EphA3-293T cells. Serum-starved EphA3-293T cells on fibronectin-coated microscopic glass coverslips were treated with clustered ephrin-A5 Fc to induce cell rounding and dynamic membrane blebbing. The video represents microscopic images taken every minute, 10 minutes before and 50 minutes after ephrin-A5 Fc stimulation, which were processed as described for Movie 1 (see Fig. 9F).

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Ephrin-A5-induced membrane blebbing of EphA3-293T cells is abrogated by the ROCK inhibitor Y-27632. Parallel cultures of serum-starved EphA3-293T cells on microscopic glass coverslips were treated, prior to stimulation with clustered ephrin-A5 Fc, for 2 hours with with 10 mM Rock inhibitor Y-27632. Although the cells still round-up upon ephrin-A5 addition, membrane blebbing is completely abrogated (see Fig. 9F)..