The novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut

Abstract

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. Here, we report a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with the Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, and stem cell overproliferation. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut.

De novo apical domain formation inside the Drosophila adult midgut epithelium

“…By contrast, more than 80% of the cells in control FRT82B MARCM clones develop a mature apical domain, while the remaining ~10% lack apical actin and are presumably ISCs or early enteroblasts (Figure 8J). Mutants in each septate junction protein disrupt the localisation of the other septate junction components, as previously reported for the embryonic and larval gut (Figure S7A-S7C)(Izumi et al ., 2021; Izumi et al ., 2016; Izumi et al ., 2012). The differences between the spectrum of phenotypes observed in mesh , Tsp2a and ssk mutants cannot therefore be explained by their distinct effects on septate junction formation and may reflect other functions of these proteins, such as the regulation of the Yki pathway(Chen et al, 2020; Izumi et al, 2019; Xu et al, 2019).…”

“…The smooth septate junctions are composed of the transmembrane proteins Mesh, Snakeskin (Ssk), Tsp2a and Hoka and the scaffolding protein, Coracle (Cora), and provide the barrier to paracellular diffusion (Furuse and Izumi, 2017; Izumi et al, 2021; Izumi et al, 2016; Izumi et al, 2012). The ISCs and early enteroblasts lie below the septate junctions between the enterocytes and only form adherens junctions with neighbouring cells, while mature enterocytes form both adherens junctions and septate junctions, as well as specialised tri-cellular junctions at the vertex between three cells.…”

An amuse-bouche of stem cell regulation: Underlying principles and mechanisms from adult Drosophila intestinal stem cells

“…In addition to tissue damage per se, diverse cellular defects in ECs cause a stress response that non-autonomously stimulates ISC proliferation (Figure 2). Functional septate junctions (SJ) are essential sensors of EC health; their perturbation leads to activation of Yki and Upd3 expression [12,[49][50][51]. Mechanistically, the SJ component, Tsp2A, promotes endocytosis and lysosomal degradation of aPKC [12].…”

Epithelial Cell Polarity During Drosophila Midgut Development

“…Endodermal epithelia form smooth septate junctions (sSJs), analogous to tight junction in mammals, which lie apical to the adherens junctions (AJs), whereas in other epithelial cells, the electron-dense AJs lie above the septate junctions, which are pleated not smooth ( Figure 1 ) ( Lane and Skaer, 1980 ; Tepass and Hartenstein, 1994b ; Baumann, 2001 ). Recent studies reveal that the smooth SJs are organised by the endoderm-specific proteins, Mesh, Snakeskin, Tsp2a and Hoka, which form a transmembrane protein complex ( Table 1 ) ( Izumi et al, 2012 , 2016 , 2021 ; Furuse and Izumi, 2017 ). The SJs at the vertices where three cells meet contain additional components, including Bark, Gli and M6, which are also found in the tri-cellular junctions in epithelia with pleated SJs ( Table 1 ) ( Schulte et al, 2003 ; Byri et al, 2015 ; Hildebrandt et al, 2015 ; Bosveld et al, 2018 ; Esmangart de Bournonville and le Borgne, 2020 ; Wittek et al, 2020 ).…”

“…The larval midgut is remarkably similar to the adult midgut at the level of cellular structure, with an apical brush border facing the gut lumen, a basal side in contact with the visceral muscle and a basal labyrinth of invaginations from the basal membrane ( Figure 3 ) ( Shanbhag and Tripathi, 2005 , 2009 ). The larval midgut also forms sSJs ( Izumi et al, 2012 , 2016 , 2021 ) and the apical domain is enriched for actin and β H- spectrin/α-spectrin, while β-spectrin/α-spectrin heterotetramers label the basolateral domain ( Dubreuil et al, 1998 ). Spectrins are not required for copper cell polarity, but loss of β H- spectrin leads to loss of the apical proton pump, the H + V-ATPase which probably causes the defect in acid secretion seen in α-spec mutant larvae ( Phillips and Thomas, 2006 ).…”