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

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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.