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Czuchra, Aleksandra (2005): Cdc42 and beta1 integrin in cell migration. Dissertation, LMU München: Faculty of Biology
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Abstract

Cell migration plays a central role in the development and maintenance of multicellular organisms. It involves regulated cell adhesion, mediated by integrins, and polarized changes of the cytoskeleton, controlled by Rho GTPases such as Cdc42. Aim of this study was to investigate the role of integrins and Cdc42 in cell migration and in particular the cross-talk between these molecules. In addition, the structure–function relationship of beta1 integrin in mediating migration associated events was studied. To test whether Cdc42 is essential for directed cell migration in mammalian cells and to investigate the cross-talks between integrin and Cdc42 mediated signalling, fibroblastoid cell lines lacking a functional Cdc42 gene were established and analyzed in wound closure assays. Contrary to the expectations, we could show that Cdc42 is neither required for integrin activation nor for integrin mediated protrusion formation. Moreover, Cdc42 has no significant influence on the speed of directed migration. However, it contributes to the directionality of migration and to the re-orientation of the Golgi apparatus into the direction of migration by a mechanism independent of Gsk3beta phosphorylation. Furthermore, we demonstrated that Cdc42 controls cell morphology, quite likely by regulating Rac1 activity. Expression of dominant negative Cdc42 (dnCdc42) in Cdc42-null cells revealed that dnCdc42 non-specifically inhibits other Rho GTPases besides Cdc42, since it aggravates the impairments observed in Cdc42-null cells, resulting in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation towards the wound. Beta1 integrins were previously shown to activate Cdc42 in response to wounding and thus to regulate the directionality of migration. We demonstrated now, that fourfold reduction of beta1 integrin expression in keratinocytes in vivo did not impair wound healing. However, keratinocyte stem cells with normal levels of beta1 integrin had a competitive advantage over the hypomorphic cells and expanded steadily in the skin of mice harbouring both cell types in the epidermis. Finally, we analysed the importance of specific amino acids of the intracellular domain of beta1 integrin in keratinocytes in vivo by generating 8 mice strains which in skin express only point or deletion mutants of beta1 integrin. Our data are for the most part strikingly different from previous results obtained in vitro and significantly revise proposed models for the role of serine and tyrosine phosphorylation and the function of a salt bridge between the integrin beta subunits and the integrin alpha tails.