The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule

Abstract

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

The septate junction protein Snakeskin is critical for epithelial barrier function and tissue homeostasis in the Malpighian tubules of adultDrosophila

“…Both SC SskRNAi and PC SskRNAi MTs also have an increased crosssectional diameter ( Figure 4D), a phenotype iterating pathological changes observed during intestinal barrier dysfunction (Izumi et al, 2019;Rera et al, 2013a;Salazar et al, 2018) and when Mesh or Tsp2A are impaired in MTs (Beyanbach et al, 2020;Jonusaite et al, 2020). Complementing these observations, while TEM scans show the septal junction span is significantly .…”

“…In keeping with this observation, Mesh/Ssk/Tsp2a do not appear to directly interact with Dlg in sSJs, as their ability to localise to junctions are independent (Yanagashi et al, 2012;Izumi et al, 2012;Izumi et al, 2016). However, the individual components of the Mesh/Ssk/Tsp2a complex are essential to appropriate localisation of Lgl (Jonusaite et al, 2020;Izumi et al, 2012;Yanagashi et al, 2012), and there exists a mutually dependent functional relationship between Lgl and Dlg (Bilder et al, 2000;2003;Izumi et al, 2012;Su et al, 2012). Dlg and Lgl, along with Scribble, comprise the 'Scribble polarity module', which acts as a regulator of cell polarity and proliferation (Bonello et al, 2019;Elsum et al, 2012;Humbert et al, 2008;2003;Khoury et al, 2020).…”

“…Failure to form sSj's properly in early development, either ubiquitously or restricted to the MTs, is lethal (Beyenbach et al, 2020;Jonusaite et al, 2020;Izumi et al, 2019;Yanagihashi et al, 2012). We therefore employed temperature-sensitive tubulin GAL80 (McGuire et al, 2004), in conjunction with either a SC-specific, c724GAL4 (Sözen et al, 1997), or PC-specific, UroGAL4…”

“…Ssk, Tsp2a, and the membrane spanning protein Mesh are mutually dependent for their localisation to, and critical for, proper formation of the sSJ's (Beyanbach et al, 2020;Jonusaite et al, 2020;Izumi et al, 2012;Izumi et al, 2016;Salazar et al, 2018;Xu et al, 2019;Yanagashi et al, 2012). This Mesh/Ssk/Tsp2a complex creates a platform for appropriate assembly and localisation of other key junctional proteins (Beyanbach et al, 2020; Jonusaite .…”

“…In invertebrates, epithelial integrity is maintained by either pleated or smooth septate junctions (pSJs and sSJs respectively), lateral intercellular contacts that are functional analogues of vertebrate tight junctions (Furuse and Izumi, 2017), with sSJs the dominant junctional complex in the MTs (Beyenbach et al, 2020;Jonusaite et al, 2020;Lane and Skaer 2016;Noirot-Timothee and Noirot, 1980). The putative cell adhesion molecule Snakeskin (Ssk), along with its counterpart Tetraspanin 2a (Tsp2a) and the membrane spanning protein Mesh are critical for the proper formation of sSJ's (Beyenbach et al, 2020;Izumi et al, 2019;2012;Jonusaite et al, 2020;Salazar et al, 2018;Xu et al, 2019;Yanagihashi et al, 2012). Mesh/Ssk/Tsp2a are also necessary, both individually and as a complex, for the appropriate localisation of associated junctional proteins such as Lethal giant larvae (Lgl) and Discs large (Dlg), both part of the Scribble polarity module (Khoury and Bilder 2020), Coracle (Cora) and Fascilin III (FasIII) (Chen et al, 2020;Izumi et al, 2012;Salazar et al, 2018;Yanagashi et al, 2012).…”