Signalling by RhoGTPases in the regulation of epithelial cell-cell adhesion and differentiation. (360G-Wellcome-084678_Z_08_Z)

Epithelial cells form polarised cellular sheets by adhering to each other via intercellular junctions. These intercellular junctions mediate adhesion but also regulate polarisation, cytoskeletal architecture and gene expression. RhoGTPases are central regulatory switches in signalling pathways. They regulate multiple cellular processes including those connected to intercellular junctions and are crucial for epithelial polarisation and differentiation. Many RhoGTPase regulators and effectors are encoded in the human genome; however, our knowledge about their functions is limited. The purpose of this proposal is to use the human epithelial cell line Caco-2 as a model to identify RhoGTPase regulators and effectors important for epithelial junction formation and differentiation, as well as to determine how such regulators cooperate to orchestrate epithelial differentiation. Our first goal is to perform a systematic analysis of all known and predicted RhoGTPase regulators and effectors usin g an RNAi library in conjunction with functional assays to monitor junction assembly, polarisation and activity of junctional signalling pathways. Our second goal is to analyse three types of regulators with opposing activities that were identified in preliminary screens to determine how they regulate junctional signalling processes and functionally interact with each other as well as known important RhoGTPase regulators to guide epithelial differentiation.