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，并调节其与环境的通讯和基质交换。它们是人类大多数实体癌症的起源。它们功能的基础是沿顶端-基底轴的严格极化组织，该组织在癌发生过程中丢失。在过去的十年中，人们发现 ESCRT 机制的功能对于果蝇翼盘上皮的完整性至关重要，果蝇是研究上皮的主要模型系统。 ESCRT 机制在跨膜蛋白 (TMP) 向溶酶体的内体运输过程中参与腔内囊泡 (ILV) 的形成，如何维持上皮极性并控制细胞增殖尚不清楚。我们开发了一种基于 RNAi 的系统，可以生成 ESCRT 核心组件 Shrub 的微弱功能丧失情况。我们发现隔膜连接处是对 ESRT 功能丧失最敏感的上皮结构。此外，Shrub/ESCRT 似乎参与了新合成的 Megatrachea (Mega) 向 SJ 的贩运。该途径还需要 Vps-Retromer 的未识别变体，该变体通常参与内体回收。因此，我们发现了 SJ 成分向顶膜的新运输途径，这对于在已建立的上皮细胞中维持 SJ 非常重要。我们的数据表明，ESCRT 调节 Mega 从基底到顶端目的地的逆转录酶依赖性运输，从而介导类似转胞吞作用的事件。在此应用中，我们将研究 Mega 和其他 SJ 组件如何运输到 SJ 以及 ESCRT 机械在此过程中的作用。此外，我们发现Shrub的耗竭会诱导EMT（上皮间充质转化）因子Snail的表达。该因子的激活已被证明可诱导肿瘤转化，因此可以解释 Shrub 功能耗竭时所见的肿瘤生长的诱导。因此，我们将了解 Snail 是如何被诱导的，以及它如何促进灌木损耗引起的肿瘤转化。在我们的分析中，我们将结合使用遗传、生化和分子工具，以及电子、共焦实时成像和超分辨率显微镜。我们的结果将有助于更深入地了解 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。此外，他们还将阐明其在上皮细胞肿瘤转化过程中的作用，从而有助于理解 EMT，这也是癌症发生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.