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

上皮细胞覆盖了我们身体的大部分表面，调节着它与环境的沟通和底物交换。它们是人类大多数实体癌症的起源。其功能的基础是沿着其顶基轴的一个严格极化的组织，该组织在癌变过程中丢失。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是必不可少的，而翼盘上皮是研究上皮细胞的主要模型系统。ESCRT机制在跨膜蛋白(TMP)内体转运到溶酶体的过程中参与管腔内小泡(ILV)的形成，但如何维持上皮极性和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核心成分灌木的弱功能丧失情况。我们发现，间隔连接是对ESCRT功能丧失最敏感的上皮结构。此外，灌木/ESCRT似乎参与了新合成的Megatrachea(Mega)向SJ的贩运。这条途径还需要一种未识别的VPS-逆转录聚合体变体，该变体通常参与内体循环。因此，我们发现了一条新的将SJ组分运输到根尖膜的途径，这对于在已经建立的上皮中维持SJ是重要的。我们的数据提示ESCRT调节MegA从底部到顶端的逆转录依赖的运输，从而介导一个类似于跨细胞的事件。在本申请中，我们将调查Mega和其他SJ组件是如何运输到SJ的，以及ESCRT机械在这一过程中扮演的角色。此外，我们还发现，灌木枯竭诱导了上皮间充质转化因子Snail的表达。这一因子的激活已被证明可以诱导肿瘤转化，因此可以解释灌木功能衰竭时肿瘤生长的诱导。因此，我们将了解蜗牛是如何被诱导的，以及它是如何在灌木枯竭引起的肿瘤转化中起作用的。在我们的分析中，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。我们的结果将使我们更深入地理解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癌变过程中的作用，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.