The function of the ESCRT machinery in the maintenance of the septate junction of Epithelia in Drosophila

上皮细胞覆盖我们身体的大部分表面，并调节其与环境的沟通和底物交换。它们是大多数人类实体癌的起源。其功能的基础是沿其顶基轴的严格极化组织，这在癌变过程中丢失。在过去的十年中，已经发现ESCRT机制的功能对于果蝇翼盘上皮的完整性至关重要，这是研究上皮的主要模型系统。在跨膜蛋白（TMPs）向溶酶体的内体运输过程中，ESCRT机制参与了腔内囊泡（ILVs）的形成，如何维持上皮极性并控制细胞增殖尚不清楚。我们开发了一个基于rnai的系统，可以生成ESCRT核心组件灌木的弱功能丧失情况。我们发现分隔连接是对ESCRT功能丧失最敏感的上皮结构。此外，灌木/ESCRT似乎参与了向SJ贩运新合成的巨毒（Mega）。这一途径还需要一种未知的Vps-Retromer变体，它通常参与内体循环。因此，我们发现了一种新的SJ成分到顶膜的运输途径，这对于已经建立的上皮中SJ的维持是重要的。我们的数据表明，ESCRT调控逆转录酶依赖的Mega从基部到其顶端的运输，从而介导类似细胞吞噬的事件。在这个应用程序中，我们将研究Mega和其他SJ组件是如何传输到SJ的，以及ESCRT机制在这个过程中的作用。此外，我们发现蜗牛上皮间充质转化因子（epithelial mesenchymal transformation factor， EMT）的表达受到灌木枯竭的诱导。该因子的激活已被证明可以诱导肿瘤转化，因此可以解释在灌木功能耗尽时诱导肿瘤生长的原因。因此，我们将发现蜗牛是如何被诱导的，以及它是如何促进灌木枯竭诱导的肿瘤转化的。对于我们的分析，我们将使用遗传，生化和分子工具的组合，以及电子，共聚焦实时成像和超分辨率显微镜。我们的研究结果将为ESCRT机制作为上皮完整性和内体运输的关键组织者的新功能提供更深入的理解。此外，它们还将阐明其在上皮肿瘤转化中的作用，从而有助于理解EMT，这也是癌变过程中的一个重要事件。

Epithelia cover most surfaces of our body and regulate its communication and substrate exchange with the environment. They are the origin of the majority of solid cancers in humans. The basis for their functionality is a strictly polarized organisation along their apico-basal axis, which is lost during cancerogenesis. In the last decade it has been found that the function of the ESCRT machinery is essential for the integrity of the wing disc epithelium of Drosophila, a major model system for studying epithelia. How the ESCRT machinery, which is involved in the formation of intraluminal vesicles (ILVs) during endosomal trafficking of transmembrane proteins (TMPs) to the lysosome, maintains epithelial polarity and controls cell proliferation is not well understood. We have developed an RNAi-based system where we can generate weak loss of function situations of the ESCRT core component Shrub. We found that the septate junction is the most sensitive epithelial structure towards loss of ESCRT function. Moreover, Shrub/ESCRT appears to be involved in the trafficking of newly synthesised Megatrachea (Mega) to the SJ. This route also requires an unidentified variant of the Vps-Retromer, which is normally involved in endosomal recycling. We therefore discovered a new trafficking route of SJ components to the apical membrane, which is important for the maintenance of SJ in an already established epithelium. Our data suggest that ESCRT regulates Retromer dependent trafficking of Mega from the basal to its apical destination, thus mediating a transcytosis like event. In this application, we will investigate how Mega and other SJ components are transported to the SJ and what the role of the ESCRT machinery is in this process. In addition, we found that the expression of the EMT (epithelial mesenchymal transformation) factor Snail is induced by the depletion of Shrub. The activation of this factor has been shown to induce neoplastic transformation and could therefore explain the induction of neoplastic growth seen upon depletion of Shrub function. We will therefore find out how Snail is induced and how it contributes to the neoplastic transformation induced by Shrub depletion.For our analysis, we will use a combination of genetic, biochemical and molecular tools, as well as electron-, confocal live imaging and super-resolution microscopy. Our results will provide a deeper understanding of a novel function of the ESCRT machinery as a key organizer of epithelial integrity and endosomal trafficking. Moreover, they will also clarify its role during neoplastic transformations of epithelia and therefore contribute to the understanding of EMT, which is also an important event during cancerogenesis.