Eph class receptors are cell surface proteins that, when activated by their ligands, cause cell-cell repulsion, which is used to limit cellular interactions, thus aiding in a variety of processes, such as tissue formation and guidance of migrating cells. Activation of one member of this family in Xenopus laevis, XEphA4, leads to reorganization of the actin cytoskeleton via a mechanism involving inhibition of the small GTPase XRhoA. The rest of the biochemical pathway by which XEphA4 mediates its effects is largely unknown. This project will test the hypothesis that the tyrosine kinase Fyn (a member of the Src family of non-receptor tyrosine kinases) is involved in signaling by XEphA4. The hypothesis will be tested using a Xenopus laevis embryo assay system. This system allows expression and activation of XEphA4 in early frog embryos (at a time when the receptor would not normally be expressed), which results in loss of cell adhesion, change in cell shape, and loss of cell polarity by epithelial cells. Candidate molecules in the signaling pathway, such as Fyn in the current project, can be tested by expressing mutant forms of the candidate molecule alone or in conjunction with XEphA4 and assessing the effect on embryo phenotype. The hypothesis will be tested using experimental approaches that comprise four objectives, each of which tests a prediction based on the hypothesis. Objective 1 will test whether constitutively-active Fyn can recreate the XEphA4 phenotype. Objective 2 will determine whether inhibition of Fyn prevents the phenotypic effects of XEphA4. Objective 3 will test whether constitutive activity of XRhoA can prevent the effects of active Fyn. Objective 4 will assay for changes in Fyn activity levels in response to XEPhA4 activity. This project should add greatly to understanding of Eph receptor signaling, and will have broader impact through training of graduate and undergraduate students, including pre-service secondary-level biology teachers, and through outreach programs to local secondary schools.
