Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fueled by mis-regulation of signaling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, we used transmission electron microscopy (TEM) on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. We find a specific requirement for Shrub in maintaining septate junction (SJ) integrity by transporting the claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery retromer. We show that ESCRT function is required for apical localization and mobility of retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent.
Keywords: D. melanogaster; ESCRT; Retromer; cell biology; claudins; developmental biology; neoplasia; septate junctions; tight junctions.
Proteins are large molecules responsible for a variety of activities that cells needs to perform to survive; from respiration to copying DNA before cells divide. To perform these roles proteins need to be transported to the correct cell compartment, or to the cell membrane. This protein trafficking depends on the endosomal system, a set of membrane compartments that can travel within the cell and act as a protein sorting hub. This system needs its own proteins to work properly. In particular, there are two sets of proteins that are crucial for the endosomal systems activity: a group of proteins known as the ESCRT (endosomal sorting complex required for transport) machinery and a complex called retromer. The retromer complex regulates recycling of receptor proteins so they can be reused, while the ESCRT machinery mediates degradation of proteins that the cell does not require anymore. In the epithelia of fruit fly larvae – the tissues that form layers of cells, usually covering an organ but also making structures like wings – defects in ESCRT activity lead to a loss of tissue integrity. This loss of tissue integrity suggests that the endosomal system might be involved in transporting proteins that form cellular junctions, the multiprotein complexes that establish contacts between cells or between a cell and the extracellular space. In arthropods such as the fruit fly, the adherens junction and the septate junction are two types of cellular junctions important for the integrity of epithelia integrity. Adherens junctions allow cells to adhere to each other, while septate junctions stop nutrient molecules, ions and water from leaking into the tissue. The role of the endosomal system in trafficking the proteins that form septate junctions remains a mystery. To better understand the role of the endosomal system in regulating cell junctions and tissue integrity, Pannen et al. blocked the activity of either the ESCRT or retromer in wing imaginal discs – the future wings – of fruit fly larvae. Pannen et al. then analyzed the effects of these endosomal defects on cellular junctions using an imaging technique called transmission electron microscopy. The results showed that both ESCRT and retromer activities are necessary for the correct delivery of septate junction components to the cell membrane. However, neither retromer nor ESCRT were required for the delivery of adherens junction proteins. These findings shed light on how retromer and the ESCRT machinery are involved in the epithelial tissue integrity of fruit fly larvae through their effects on cell junctions. Humans have their own versions of the ESCRT, retromer, and cell junction proteins, all of which are very similar to their fly counterparts. Since defects in the human versions of these proteins have been associated with a variety of diseases, from infections to cancer, these results may have implications for research into treating those diseases.
© 2020, Pannen et al.