Investigation into how Cdc42 is activated to define the apical domain of epithelial cells in Drosophila

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Theme: World-Class Underpinning BioscienceThe small GTPase Cdc42 is a highly conserved key regulator of apicobasal polarity. It plays an important role in localising apical determinants, such as Par6 and aPKC, and tight control of its activity is crucial for establishment and maintenance of epithelial polarity. Activity of Cdc42 is controlled by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that promote activation or inactivation, respectively. Our lab has identified putative Cdc42 GEFs in Drosophila, including a GEF that phenocopies Cdc42 mutants in the follicular epithelium. In this project I would like to examine the activity of this GEF, CG42674, on Cdc42 and other members of the Rho GTPase family. In addition, I want to determine if the GEF is localised to the apical domain in follicle cells, where active Cdc42 (Cdc42-GTP) is found. I also want to take an unbiased approach to elucidate how CG42674 is localised. It would be interesting to identify the protein responsible for localising the GEF as it is upstream of Cdc42, a master regulator of polarity. To study this question, I will make use of novel biotin-based proximity labelling technique TurboID. This will be the first time that TurboID is used to answer a biological question in Drosophila. I would also like to examine the role that other putative Cdc42 GEFs play in polarity maintenance of various epithelia. Using TurboID I could identify potential interacting proteins for these other GEFs in their relevant tissues and discover how they are localised to the correct domain where they can activate Cdc42. I aim to find common mechanisms across various epithelia in how the apical domain is maintained through localised activity of the relevant dc42 GEF.