Objective Rho-family GTPases control many aspects of cell behavior through the regulation of multiple signaling pathways. Failure to properly regulate Rho GTPases can have deleterious consequences in human development and disease. A key group of spatial and temporal regulators of Rho-family GTPases are the Rho GEF proteins. Rho GEFs serve to couple diverse regulatory stimuli to Rho GTPase activation. There are a large number of Rho GEFs encoded by the human genome and a systematic study addressing cellular function is needed. With this proposal, I aim to complete an unbiased large-scale functional analysis of all the Rho GEF proteins using a lentiviral-RNAi based strategy to identify Rho-family GEF proteins involved in epithelial cell morphogenesis. To achieve this project I will use the three-dimensional Madin-Darby canine kidney epithelial cell (3D-MDCK) system, which is an excellent model to investigate epithelial morphogenesis in vitro. I will verify RNAi mediated knockdown of expression for candidate GEFs and demonstrate that the phenotypes caused by RNAi are not primarily due to off-target effects. Then, I will characterize the phenotypes of Rho GEF RNAi knockdowns in detail and determine the subcellular localization of the GEF proteins. Finally, I aim to determine the Rho GTPase specificity for Rho GEF candidates. The analysis of Rho GEF function in the MDCK 3D cell culture system might have particular relevance to cancer since their aberrant regulation can lead to malignant transformation and a majority of human tumors are thought to be epithelial in origin. In this proposal, I will carry out a more systematic, genome wide knockdown of all known human Rho GEFs with the goal of identifying and characterizing important regulators of MDCK epithelial cell morphogenesis. I hope that the insights I gain from the analysis of Rho GEF function in MDCK epithelial cells will aid our understanding of development and disease in both the kidney and other epithelial tissues. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsmedical and health sciencesclinical medicineoncologynatural sciencesmathematicspure mathematicsmathematical analysisfunctional analysisnatural sciencesbiological sciencesgeneticsgenomes Keywords Epithelial morphogenesis Health sciences RNAi screening Rho GTPases RhoGEFs cell polarity Programme(s) FP7-PEOPLE - Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013) Topic(s) PEOPLE-2007-4-3.IRG - Marie Curie Action: "International Reintegration Grants" Call for proposal FP7-PEOPLE-2007-4-3-IRG See other projects for this call Funding Scheme MC-IRG - International Re-integration Grants (IRG) Coordinator AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS EU contribution € 100 000,00 Address CALLE SERRANO 117 28006 Madrid Spain See on map Region Comunidad de Madrid Comunidad de Madrid Madrid Activity type Research Organisations Administrative Contact Mar García-Ferrer (Ms.) Links Contact the organisation Opens in new window Website Opens in new window Total cost No data
