Dynamics of epithelial polarity proteins in Drosophila

描述（由申请人提供）：已经确定了许多指导上皮片内极性建立的分子。然而，我们对这些蛋白质如何定位于细胞内特定位置的理解存在一个关键的空白。考虑到这一点，本应用程序的目的是建立一套工具和技术来解决上皮极性蛋白的动态定位。迄今为止，大多数果蝇极性蛋白已经在固定组织制剂的稳态条件下进行了检测。这项工作的重点是利用可光切换的荧光蛋白来标记已被证明可以直接极化细胞行为的关键极性蛋白。为此，这些研究将在果蝇胚胎中建立的实时成像条件下检查两种最有希望的光开关蛋白，EosFP和Dronpa。在评估了EosFP和Dronpa在果蝇胚胎中的行为后，将建立携带两种关键上皮极性蛋白光开关融合的转基因系。这将允许，第一次，在体内测量这些极性蛋白的周转和动态定位。这项工作中使用的方法是创新的，因为它将利用最前沿的成像技术，这些技术刚刚开始应用于果蝇，以建立指导上皮极性的蛋白质复合物的稳定性。这项工作的意义在于，这些相同极性的蛋白质在正常人类发育和体内平衡所需的上皮过程中具有重要的功能保护作用。

DESCRIPTION (Provided by Applicant): Many molecules that direct the establishment of polarities within epithelial sheets have been identified. However, there is a critical gap in our understanding of how these proteins become localized to specific places within the cell. With this in mind, the objective of this application is to establish a set of tools and techniques to address the dynamic localization of epithelial polarity proteins. To date, most polarity proteins in Drosophila have been examined under steady-state conditions in fixed tissue preparations. The focus of the proposed work is to take advantage of photoswitchable fluorescent proteins to tag crucial polarity proteins that have been demonstrated to direct polarized cell behaviors. To this end, these studies will examine two of the most promising photoswitchable proteins, EosFP and Dronpa, under established live imaging conditions in the Drosophila embryo. After evaluating the behavior of EosFP and Dronpa in the fly embryo, transgenic lines carrying photoswitchable fusions to two critical epithelial polarity proteins will be established. This will allow, for the first time, the in vivo measurement of the turnover and dynamic localization of these polarity proteins. The approach used in this work is innovative because it will draw on cutting edge imaging techniques that are just beginning to be applied to Drosophila to establish the stability of the protein complexes that guide epithelial polarity. The significance of this work derives from the important functional conservation of these same polarity proteins in epithelial processes required for normal human development and homeostasis. PROJECT NARRATIVE: Epithelial tissues, such as the human skin and gut, maintain an essential polarity that allows the cells in these tissues to confront both the external environment of the organism and the aqueous internal environment. The maintenance of this polarity is essential to the barrier function of epithelia, and the loss of this polarity is associated with the metastasis of many epithelial cancers. The intention of this proposed study is to examine the dynamics by which polarity proteins are localized to specific places in the cell, thus leading to a better understanding of how the critical polarized function of epithelial tissues is maintained and can be subverted by disease.