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

主题：世界级支撑生物科学小GTP酶CDC42是根尖基极性的高度保守的关键调节因子。它在顶端决定因素，如Par6和aPKC的定位中起着重要作用，严格控制其活性对于建立和维持上皮极性至关重要。Cdc42的活性分别由鸟嘌呤核苷酸交换因子(GEF)和GTP酶激活蛋白(GAP)控制，它们分别促进激活或失活。我们的实验室已经在果蝇中鉴定出推定的CDC42GEF，包括一种在滤泡上皮中表型复制CDC42突变体的环境基金。在这个项目中，我想研究全球环境基金CG42674对CDC42和Rho GTPase家族其他成员的活动。此外，我还想确定该蛋白是否定位于卵泡细胞的顶端区域，在那里发现了活性的CDC42(CDC42-GTP)。我还想采取一种公正的方法来阐明CG42674是如何本地化的。确定负责定位全球环境基金的蛋白质将是有趣的，因为它位于极性的主要调节因子CDC42的上游。为了研究这个问题，我将利用新的基于生物素的邻近标记技术TurboID。这将是TurboID首次用于回答果蝇的生物学问题。我还想研究其他推定的CDC42GEF在各种上皮细胞的极性维持中所起的作用。使用TurboID I可以在相关组织中识别这些其他GEF的潜在相互作用蛋白，并发现它们如何定位到正确的区域，在那里它们可以激活CDC42。我的目标是在不同的上皮细胞中寻找共同的机制，了解如何通过相关的DC42全球环境基金的局部活动来维持顶端区域。

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.