Abstract
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.
Highlights
Developmental and physiological functions of epithelia rely on a set of cellular junctions, linking cells within the tissue to a functional unit
By dissecting the intracellular trafficking itinerary of the Claudin Megatrachea, we reveal that biosynthetic delivery of this core SJ component depends on a complex basal to apical transcytosis route relying on Endosomal Sorting Complex Required for Transport (ESCRT) and Retromer functions. 152 Results 153 154 ESCRT knockdown affects SJ integrity To analyse the impact of ESCRT loss of function on junctional integrity, transmission electron microscopy (TEM) was used on wing imaginal discs that have been depleted of ESCRT function
We failed to find any of these proteins to be required for the regulation of Mega membrane levels, raising questions on the mechanism governing cargo-selective complex (CSC) dependent transport of Mega (Figure 2-figure supplement 5). based on above findings, we propose a novel function of the Retromer CSC in delivery of SJ core components to the junction, thereby contributing to SJ homeostasis in the proliferative wing disc epithelium. 311 ESCRT regulates subcellular localization and mobility of the Retromer CSC The above results indicate that Retromer is required for regulating membrane levels of Mega and other SJ components
Summary
Developmental and physiological functions of epithelia rely on a set of cellular junctions, linking cells within the tissue to a functional unit. Proteins of the conserved Claudin family play a key role in establishing and regulating TJ permeability in the intercellular space by homo- and heterophilic interactions with Claudins of neighbouring cells (Gunzel and Yu, 2013) Arthropods, such as Drosophila, do not possess TJs but a functionally similar structure in ectoderm-derived epithelia termed pleated Septate Junction (pSJ, SJ hereafter), characterized by protein dense septa lining the intercellular space in electron micrographs (Gilula et al, 1970). While junction formation during embryogenesis requires the SJ localized cytoplasmic protein Dlg, this basolateral cell polarity factor is not a structural part of the immobile junction core complex (Oshima and Fehon, 2011; Woods et al, 1996) This explains the functional separation of barrier formation and apicobasal polarity despite the close association of Dlg-complex components with the SJ. SJ components are frequently associated with endosomal compartments, suggesting a role for the endosomal system in coordinating transport and turnover of SJ complexes (Nilton et al, 2010; Tempesta et al, 2017; Tiklova et al, 2010)
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