InteractiveFly: GeneBrief

Snakeskin: Biological Overview | References

BIOLOGICAL OVERVIEW

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. This study reports 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 (Izumi, 2021).

Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, occluding junctions act as barriers that control the free diffusion of solutes through the paracellular pathway. Septate junctions (SJs) are occluding junctions in invertebrates and form circumferential belts along the apicolateral region of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectoderm-derived epithelia and surface glia surrounding the nerve cord, whereas sSJs are found mainly in the endoderm-derived epithelia, such as the midgut and gastric caeca. Despite being derived from the ectoderm, the outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs. Furthermore, pSJs and sSJs are distinguished by the arrangement of septa. For example, the septa of pSJs form regular undulating rows, wherea1 those in sSJs form regularly spaced parallel lines in the oblique sections in lanthanum-treated preparations. To date, more than 20 pSJ-related proteins have been identified and characterized in Drosophila. In contrast, only three membrane-spanning proteins, Ssk, Mesh and Tsp2A, have been reported as specific molecular constituents of sSJs (sSJ proteins) in Drosophila (Furuse, 2017). Therefore, the mechanisms underlying sSJ organization and the functional properties of sSJs remain poorly understood compared with pSJs. Ssk has four membrane-spanning domains; two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop (Yanagihashi, 2012). Mesh is a cell-cell adhesion molecule, which has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain and a sushi domain (Izumi, 2012). Tsp2A is a member of the tetraspanin family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn (Izumi, 2016). The loss of ssk, mesh and Tsp2A causes defects in the ultrastructure of sSJs and the barrier function against a 10 kDa fluorescent tracer in the Drosophila larval midgut. Ssk, Mesh and Tsp2A interact physically and are mutually dependent for their sSJ qlocalization (Izumi, 2012; Izumi, 2016). Thus, Ssk, Mesh and Tsp2A act together to regulate the formation and barrier function of sSJs. Furthermore, Ssk, Mesh and Tsp2A are localized in the epithelial cell-cell contact regions in the Drosophila Malpighian tubules in which sSJs are present. Recent studies have shown that the knockdown of mesh and Tsp2A in the epithelium of Malpighian tubules leads to defects in epithelial morphogenesis, tubule transepithelial fluid and ion transport, and paracellular macromolecule permeability in the tubules (Jonusaite, 2020; Beyenbach, 2020). Thus, sSJ proteins are involved in the development and maintenance of functional Malpighian tubules in Drosophila (Izumi, 2021).

The Drosophila adult midgut consists of a pseudostratified epithelium, which is composed of absorptive enterocytes (ECs), secretory enteroendocrine cells (EEs), intestinal stem cells (ISCs), EC progenitors (enteroblasts: EBs) and EE progenitors (enteroendocrine mother cells: EMCs). The sSJs are formed between adjacent ECs and between ECs and EEs. To maintain midgut homeostasis, ECs and EEs are continuously renewed by proliferation and differentiation of the ISC lineage through the production of intermediate differentiating cells, EBs and EMCs. Recently, it has been reported that the knockdown of sSJ proteins Ssk, Mesh and Tsp2A in the midgut causes intestinal hypertrophy accompanied by the overproliferation of ECs and ISC. These results indicate that sSJs play a crucial role in maintaining tissue homeostasis through the regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut. Furthermore, Chen (2018) reported that the loss of mesh and Tsp2A in adult midgut epithelial cells causes defects in cellular polarization, although no remarkable defects in epithelial polarity were found in the first-instar larval midgut cells of ssk, mesh and Tsp2A mutants. Thus, sSJs may contribute to the establishment of epithelial polarity in the adult midgut (Izumi, 2021).

During the regeneration of the Drosophila adult midgut epithelium, various signaling pathways are involved in the proliferation and differentiation of the ISC lineage. Atypical protein kinase C (aPKC) is an evolutionarily conserved key determinant of apical-basal epithelial polarity (Ohno, 2015). Importantly, Chen (2018) have reported that aPKC is dispensable for the establishment of epithelial cell polarity in the Drosophila adult midgut. It has been reported that aPKC is required for differentiation of the ISC linage in the midgut. The Hippo signaling pathway is involved in maintaining tissue homeostasis in various organs. In the Drosophila midgut, inhibition of the Hippo signaling pathway activates the transcriptional co-activator Yorkie (Yki), which results in accelerated ISC proliferation via the Unpaired (Upd)-Jak-Stat signaling pathway. Recent studies have shown that Yki is involved in ISC overproliferation caused by the depletion of sSJ proteins in the midgut. Furthermore, Xu (2019) showed that aPKC is activated in the Tsp2A-RNAi-treated midgut, leading to activation of its downstream target Yki and causing ISC overproliferation through the activation of the Upd-Jak-Stat signaling pathway. Thus, crosstalk between aPKC and the Hippo signaling pathways appears to be involved in ISC overproliferation caused by Tsp2A depletion (Izumi, 2021).

To further understand the molecular mechanisms underlying sSJ organization, a deficiency screen was performed for Mesh localization, and the integral membrane protein Hoka was identified as a novel component of Drosophila sSJs. Hoka consists of a short extracellular region and the characteristic repeating 4-amino acid motifs in the cytoplasmic region, and is required for the organization of sSJ structure in the midgut. Hoka and Ssk, Mesh, and Tsp2A show interdependent localization at sSJs and form a complex with each other. The knockdown of hoka in the adult midgut results in intestinal barrier dysfunction, aPKC- and Yki-dependent ISC overproliferation, and epithelial tumors. Thus, Hoka plays an important role in sSJ organization and in maintaining ISC homeostasis in the Drosophila midgut (Izumi, 2021).

The identification of Ssk, Mesh and Tsp2A has provided an experimental system to analyze the role of sSJs in the Drosophila midgut (Furuse, 2017). Recent studies have shown that sSJs regulate the epithelial barrier function and also ISC proliferation and EC behavior in the midgut (Salazar, 2018; Xu, 2019; Izumi, 2019; Chen, 2020). Furthermore, sSJs are involved in epithelial morphogenesis, fluid transport and macromolecule permeability in the Malpighian tubules (Jonusaite, 2020; Beyenbach, 2020). This study reports the identification of a novel sSJ-associated membrane protein Hoka. Hoka is required for the efficient accumulation of other sSJ proteins at sSJs and the correct organization of sSJ structure. The knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, increased ISC proliferation mediated by aPKC and Yki activities, and epithelial tumors. Thus, Hoka contributes to sSJ organization and the maintenance of ISC homeostasis in the Drosophila midgut (Izumi, 2021).

Arthropod sSJs have been classified together based on their morphological similarity. The identification of sSJ proteins in Drosophila has provided an opportunity to investigate whether sSJs in various arthropod species share similarities at the molecular level. However, Hoka homolog proteins appear to be conserved only in insects upon a database search, suggesting compositional variations in arthropod sSJs (Izumi, 2021).

Interestingly, the cytoplasmic region of Hoka includes three YTPA motifs. The same or similar amino acid motifs are also present in the Hoka homologs of other holometabolous insects, such as other Drosophila species, the mosquito, beetle (YTPA motif), butterfly, ant, bee, sawfly, moth (YQPA motif) and flea (YTAA motif), although the number of these motif(s) vary (1 to 3 in Drosophila species, 1 in other holometabolous insects). In contrast, the motif is not present in hemimetabolous insects. The extensive conservation of the YTPA/YQPA/YTAA motif in holometabolous insects suggests that the motif was evolutionarily acquired and plays a critical role in the molecular function of Hoka. It would be interesting to investigate the role of the YTPA/YQPA/YTAA motif in sSJ organization of holometabolous insects (Izumi, 2021).

The extracellular region of Hoka appears to be composed of 13 amino acids alone after the cleavage of the signal peptide, which is too short to bridge the 15-20 nm intercellular space of sSJs. Thus, Hoka is unlikely to act as a cell adhesion molecule in sSJs. Indeed, the overexpression of Hoka-GFP in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2021).

The loss of an sSJ protein results in the mislocalization of other sSJ proteins, indicating that sSJ proteins are mutually dependent for their sSJ localization. In thessk -deficient midgut, Mesh and Tsp2A were distributed diffusely in the cytoplasm (Izumi, 2012, 2016). In the mesh mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Tsp2A was distributed diffusely in the cytoplasm (Izumi, 2012, 2016). In the Tsp2A-mutant midgut, Ssk was localized at the apical and lateral membranes, whereas Mesh was distributed diffusely in the cytoplasm (Izumi, 2016). Among these three mutants, the mislocalization of Ssk, Mesh or Tsp2A is consistent; Mesh and Tsp2A were distributed in the cytoplasm, whereas Ssk was localized at the apical and lateral membranes. However, in the hoka-mutant larval midgut, Mesh and Tsp2A were distributed along the lateral membrane, whereas Ssk was mislocalized to the apical and lateral membranes. Interestingly, in some hoka mutant midguts, Ssk, Mesh and Tsp2A were localized to the apicolateral region, as observed in the wild-type midgut. Differences in subcellular misdistribution of sSJ proteins between the hoka mutant and the ssk, mesh and Tsp2A-mutants indicate that the role of Hoka in the process of sSJ formation is different from that of Ssk, Mesh or Tsp2A. Ssk, Mesh and Tsp2A may form the core complex of sSJs, and these proteins are indispensable for the generation of sSJs, whereas Hoka facilitates the arrangement of the primordial sSJs at the correct position, i.e. the apicolateral region. This Hoka function may also be important for rapid paracellular barrier repair during the epithelial cell turnover in the adult midgut. Notably, during the sSJ formation process of the outer epithelial layer of the proventriculus (OELP, the sSJ targeting property of Hoka was similar to that of Mesh, implying that Hoka may have a close relationship with Mesh, rather than Ssk and Tsp2A during sSJ development (Izumi, 2021).

The knockdown of hoka in the adult midgut leads to a shortened lifespan in adult flies, intestinal barrier dysfunction, increased ISC proliferation and the accumulation of ECs. These results are consistent with the recent observation for ssk, mesh and Tsp2A-RNAi in the adult midgut. The intestinal barrier dysfunction caused by RNAi for sSJ proteins may permit the leakage of particular substances from the midgut lumen, which may induce particular cells to secrete cytokines and growth factors for ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs. In the latter case, Xu (2019) showed that Tsp2A knockdown in ISCs/EBs or ECs hampers the endocytic degradation of aPKC, thereby activating the aPKC and Yki signaling pathways, leading to ISC overproliferation in the midgut. Therefore, Xu (2019) proposed that sSJs are directly involved in the regulation of aPKC and the Hippo pathway-mediated intracellular signaling for ISC proliferation. This study has shown that the expression of hoka-RNAi together with aPKC-RNAi or yki-RNAi in ECs significantly reduced ISC overproliferation caused by hoka-RNAi. Thus, aPKC- and Yki-mediated ISC overproliferation appears to commonly occur in sSJ protein-deficient midguts. However, the possibility that the leakage of particular substances through the paracellular route may be involved in ISC overproliferation in the sSJ proteins-deficient midgut cannot be excluded (Izumi, 2021).

It has been reported that apical aPKC staining is observed in ISCs but is barely detectable in ECs. This study found that the expression of hoka-RNAi in ECs increased aPKC staining in the midgut. Additionally, in the hoka-RNAi midgut, apical aPKC staining was observed in ISCs and in differentiated cells, including EC-like cells. Thus, apical and increased cytoplasmic aPKC may contribute to ISC overproliferation. Interestingly, EC-like cells in the hoka-RNAi midgut do not always localize aPKC to the apical regions. Apical aPKC staining was detected in EC-like cells mounted by other cells but was barely detectable in the lumen-facing EC-like cells. These mounted cells are thought to be newly generated cells after the induction of hoka-RNAi, which may not be able to exclude aPKC from the apical region in the crowded cellular environment. A recent study showed that aberrant sSJ formation caused by Tsp2A-depletion impairs aPKC endocytosis and increases aPKC localization in the membrane of cell borders (Xu, 2019). The sSJ proteins, including Hoka, may also regulate endocytosis to exclude aPKC from the apical membrane of ECs. The identification of molecules involved in aPKC-mediated ISC proliferation may provide a better understanding of the aPKC-mediated signaling pathway, as well as the mechanisms underlying the increased expression and apical targeting of aPKC in the ECs deficient for sSJ proteins (Izumi, 2021).

The Snakeskin-Mesh complex of smooth septate junction restricts Yorkie to regulate intestinal homeostasis in Drosophila

Tight junctions in mammals and septate junctions in insects are essential for epithelial integrity. This study shows that, in the Drosophila intestine, smooth septate junction proteins provide barrier and signaling functions. During an RNAi screen for genes that regulate adult midgut tissue growth, loss of two smooth septate junction components, Snakeskin and Mesh, were found to cause a hyperproliferation phenotype. By examining epitope-tagged endogenous Snakeskin and Mesh, this study demonstrated that the two proteins are present in the cytoplasm of differentiating enteroblasts and in cytoplasm and septate junctions of mature enterocytes. In both enteroblasts and enterocytes, loss of Snakeskin and Mesh causes Yorkie-dependent expression of the JAK-STAT pathway ligand Upd3, which in turn promotes proliferation of intestinal stem cells. Snakeskin and Mesh form a complex with each other, with other septate junction proteins and with Yorkie. Therefore, the Snakeskin-Mesh complex has both barrier and signaling function to maintain stem cell-mediated tissue homeostasis and (Chen, 2020).

This study used knockin and knockout alleles of Ssk and Mesh to demonstrate their functions in both EBs and ECs to regulate ISC proliferation. Ssk and Mesh have low but detectable expression in the cytoplasm of EBs, while that in ECs is mainly in septate junctions but also with some cytoplasmic localization. Multiple lines of evidence suggest that the Ssk and Mesh expression in EBs is of functional importance. First, the EB driver Su(H) > has expression in fewer cells when compared with the Myo1A > driver but still can cause comparable Upd3 expression and proliferation phenotypes. Second, mutant Ssk and mesh MARCM clones have detectable upd3-promoter-LacZ reporter expression in late EBs, and in addition to that in mature ECs. Third, the Su(H) > Ssk or mesh RNAi had very minor Smurf and lethality phenotype, suggesting that the EB RNAi effects do not linger into mature ECs, but yet can cause strong proliferation phenotypes (Chen, 2020).

Recent reports have expanded the Hpo/Mst pathway to include Misshapen (Msn) and Happyhour (Hppy), as well as their mammalian homologs MAP4K1-7. Many membrane-associated proteins, such as cadherin-like protein FAT, adherens junction protein α-catenin, and tight junction protein Angiomotin are involved in the Hpo/Mst pathway by regulating upstream components. The current results suggest that smooth septate junctions may act as a signaling platform by directly binding to Yki. That previous protein interaction screens conducted in S2 cells had not identified the Yki complex with Ssk or Mesh may be because Ssk and Mesh are expressed much more highly in the gut than in other tissues (Chen, 2020).

Another smooth septate junction component Tsp2A regulates midgut homeostasis through the aPKC-Hpo pathway, possibly involving endosomal trafficking. This study also observed that Ssk and Mesh had detectable expression in cytoplasmic punctae. However, Tsp2A can act in the whole ISC-EB-EC lineage (Xu, 2019), while this study did not observe a function of Ssk and Mesh in early EBs. Therefore, it is possible that Mesh, Ssk, and Tsp2A can form a complex and are components of the smooth septate junction but each may also have independent functions (Chen, 2020).

Disruption of paracellular junctions in adult midgut leads to epithelial disorganization along the digestive track. Loss of tricellular junction protein Gliotactin leads to activation of the JNK pathway in ECs to stimulate ISC proliferation. Prolonged RNAi of Ssk and mesh, especially in ECs, leads to leaky gut and lethality, consistent with loss of septate junction integrity. The depletion of Yki alone after longer RNAi of Ssk or mesh in ECs, however, is not sufficient to suppress all the phenotypes, suggesting that such stress may stimulate multiple downstream response pathways. Regarding Yki target genes, upd3 is the best-characterized target in the midgut. The other well-known targets from imaginal discs, including DIAP1 and Bantam, are not good targets for Yki in the adult midgut. Meanwhile, one report shows that ImpL2 expression is highly increased in response to over-activated Yki in the midgut, and regulates tissue metabolism. The physiological stimulation of ImpL2 by Yki is still unclear. This study has also assayed for ImpL2 in the midgut after Ssk or mesh RNAi, but an increased expression of ImpL2 was not observed. It is speculated that ImpL2 may be a direct or indirect target related to metabolic phenotype induced by cancer-promoting genes, and therefore may not be regulated by the septate junction proteins Ssk or Mesh. Gut leakage may drive inflammation and trigger systematic immune response contributed by hemocytes, which in turn trigger ISC division indirectly. Further investigation will provide a more complete picture of how different pathways are involved (Chen, 2020).

Septate junctions regulate gut homeostasis through regulation of stem cell proliferation and enterocyte behavior in Drosophila

Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route of epithelial cells in the Drosophila midgut. Previous work identified three sSJ-associated membrane proteins, Ssk, Mesh, and Tsp2A and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. This study investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ-proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ-protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAP kinase and Jak-Stat pathways in stem cells. Loss of cytokines Unpaired2 and Unpaired3, which are involved in Jak-Stat pathway activation, reduced the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut (Izumi, 2019).

In the Drosophila midgut epithelium, the paracellular barrier is mediated by specialized cell-cell junctions known as sSJs. Previous studies revealed that three sSJ-associated membrane proteins, Ssk, Mesh and Tsp2A, are essential for the organization and function of sSJs. In this study, the sSJ proteins were depleted from ECs in the Drosophila adult midgut; they were also required for the barrier function in the adult midgut epithelium. Interestingly, the reduced expression of sSJ proteins in ECs led to remarkably shortened lifespan in adult flies, increased ISC proliferation and intestinal hypertrophy accompanied by accumulation of morphologically aberrant ECs in the midgut. The intestinal hypertrophy caused by mesh depletion was reduced by loss of upd2 and upd3 without profound suppression of ISC proliferation, without recovery of the shortened lifespan, and without recovery of the midgut barrier dysfunction. It also found that Tsp2A mutant clones promoted ISC proliferation in a non-cell-autonomous manner. Taken together, it is propose that sSJs play a crucial role in maintaining tissue homeostasis through regulation of ISC proliferation and EC behavior in the Drosophila adult midgut. The adult Drosophila intestine provides a powerful model to investigate the molecular mechanisms behind the emergence and progression of intestinal metaplasia and dysplasia, which are associated with gastrointestinal carcinogenesis in mammals. Given that Drosophila intestinal dysplasia is associated with over-proliferation of ISCs and their abnormal differentiation, the intestinal hypertrophy observed in the present study should be categorized as a typical dysplasia in the Drosophila intestine. In this study, depletion of sSJ proteins from the ECs was mostly performed through RNAi, which may raise a concern about off-target effects. Nevertheless, it is safe to say that the midgut phenotypes were derived from specific effects of sSJ protein depletion because the essentially same phenotypes were observed in the RNAi lines for different sSJ proteins and additional RNAi lines for mesh and Tsp2A (Izumi, 2019).

Based on these observations, the following scenario is hypothesized for the hypertrophy generation in the sSJ-protein-deficient midgut. First, depletion of sSJ proteins from enterocytes (ECs) leads to disruption of sSJs in the midgut. Second, the impaired midgut barrier function caused by disruption of sSJs results in leakage of harmful substances from the intestinal lumen, thereby inducing the expression of cytokines and growth factors, such as Upd and EGF ligands, in the midgut. Alternatively, disruption of sSJs causes direct activation of a particular signaling pathway that induces expression of cytokines and growth factors by ECs. Third, proliferation of ISCs is promoted by activation of the Jak-Stat and Ras-MAPK pathways. Fourth, EBs produced by the asymmetric division of ISCs differentiate into ECs with impaired sSJs in response to cytokines such as Upd2 and/or Upd3. Consistent with this scenario, increased mRNA expression of upd3 in the ssk- and Tsp2A-deficient midgut has been reported very recently (Salazar, 2018; Xu, 2019). Finally, the ECs fail to integrate into the epithelial layer and thus become stratified in the midgut lumen to generate hypertrophy. Interestingly, loss of upd2 and upd3 reduced the intestinal hypertrophy caused by depletion of mesh, but not the increased ISC proliferation. These findings imply that Upd2 and/or Upd3 preferentially promote enteroblast (EB) differentiation rather than intestinal stem cell (ISC) proliferation. Considering that Upd-Jak-Stat signaling is required for both ISC proliferation and EB differentiation, Upd2 and/or Upd3 may predominantly promote EB differentiation and accumulation of ECs, while other cytokines such as Upd1 and/or EGF ligands may activate ISC proliferation in the sSJ-disrupted midgut. Meanwhile, Upd-Jak-Stat signaling-mediated ISC proliferation also occurs during experimental induction of apoptosis of ECs. Given that apoptosis of ECs is thought to cause the disruption of epithelial integrity followed by midgut barrier dysfunction, it may induce ISC proliferation by the same mechanism as that observed in the sSJ-protein-deficient midgut. Thus, a possibility cannot be excluded that depletion of sSJ proteins initially causes apoptosis of ECs and thereby leads to increased ISC proliferation. This possibility needs to be examined in future studies. Interestingly, the ISC proliferation induced by the loss of sSJ proteins was non-cell-autonomous. Mechanistically, disruption of the intestinal barrier function caused by impaired sSJs may permit the leakage of particular substances from the midgut lumen, which would induce particular cells to secrete cytokines and growth factors, such as Upd and EGF ligands, and stimulate ISC proliferation. Alternatively, sSJs or sSJ-associated proteins may be directly involved in the secretion of cytokines and growth factors through the regulation of intracellular signaling in the ECs (Izumi, 2019).

In this study,abnormal morphology and aberrant F-actin and Dlg distributions were observ ed in ssk-, mesh- and Tsp2A-RNAi ECs. Consistent with these results, Chen (2018) recently reported that loss of mesh and Tsp2A in clones causes defects in polarization and integration of ECs in the adult midgut. In contrast, no remarkable defects in the organization and polarity of ECs were observed in the ssk-, mesh- and Tsp2A-mutant midgut in first-instar larvae, suggesting that sSJ proteins are not required for establishment of the initial epithelial apical-basal polarity. This discrepancy may be explained by the marked difference between the larval and adult midguts: ECs in the larval midgut are postmitotic, while those in the adult midgut are capable of regeneration by the stem cell system. In the sSJ protein-deficient adult midgut, activated proliferation of ISCs generates excessive ECs. These ECs may lack sufficient cell-cell adhesion, because of impaired sSJs, fail to become integrated into the epithelial layer and detach from the basement membrane, leading to loss of normal polarity. Because sSJs seem to be the sole continuous intercellular contacts between adjacent epithelial cells in the midgut, it is reasonable to speculate that sSJ-disrupted ECs have a reduced ability to adhere to other cells (Izumi, 2019).

A recent study revealed that depletion of the tricellular junction protein Gliotactin from ECs leads to epithelial barrier dysfunction, increased ISC proliferation and blockade of differentiation in the midgut of young adult flies (Resnik-Docampo, 2017). In contrast to the findings after depletion of sSJ proteins presented in this study, the gliotactin-deficient flies did not appear to exhibit intestinal hypertrophy accompanied by accumulation of ECs throughout the midgut. Furthermore, the lifespan of gliotactin-deficient flies was found to be longer than that found for sSJ-protein-deficient flies. The difference in phenotypes found between the Resnik-Docampo (2017) study and the present study may reflect the difference in the degrees of sSJ deficiency - disruption of entire bicellular sSJs or tricellular sSJs only. Aging has also been reported to be correlated with barrier dysfunction, increased ISC proliferation and accumulation of aberrant cells in the adult midgut. The hypertrophy formation in the sSJ-disrupted midgut accompanied by increased ISC proliferation and accumulation of aberrant ECs raise the possibility that disruption of sSJs is the primary cause of the alterations in the midgut epithelium with aging (Izumi, 2019).

During the preparation of this manuscript, two groups published interesting phenotypes of the sSJ-protein-deficient adult midgut in Drosophila that are highly related to the present study. Salazar (2018) reported that reduced expression of ssk in ECs leads to gut barrier dysfunction, altered gut morphology, increased stem cell proliferation, dysbiosis and reduced lifespan. That study also showed that upregulation of Ssk in the midgut protects flies against microbial translocation, limits dysbiosis and prolongs lifespan. Meanwhile, Xu (2019) reported that depletion of Tsp2A from ISCs and EBs causes accumulation of ISCs and EBs and a swollen midgut with multilayered epithelium, similar to the current observations. They also showed that knockdown of ssk and mesh in ISCs and EBs results in accumulation of ISCs and EBs. Importantly, that study demonstrated that Tsp2A depletion from ISCs and EBs causes excessive aPKC-Yki-JAK-Stat activity and leads to increased stem cell proliferation in the midgut. That study further showed that Tsp2A is involved in endocytic degradation of aPKC, which antagonizes the Hippo pathway. Those results strongly suggest that sSJs are directly involved in the regulation of intracellular signaling for ISC proliferation. In that study, Tsp2A knockdown in ISCs and EBs caused no defects in the midgut barrier function, in contrast to what is shown in the present study. This discrepancy may be due to differences in the GAL4 drivers used in each study or the conditions for the barrier integrity assay (Smurf assay). In addition, Xu (2019) mentioned that MARCM clones generated from ISCs expressing Tsp2A-RNAi grow much larger than control clones, while this study found no remarkable size difference between Tsp2A-mutant clones and control clones. Such discrepancies need to be reconciled by future investigations. To further clarify the mechanistic details for the role of sSJs in stem cell proliferation, it will be interesting to analyze the effects of sSJ protein depletion on the behavior of adult Malpighian tubules, which also have sSJs and a stem cell system (Izumi, 2019).

This study has demonstrated that sSJs play a crucial role in maintaining tissue homeostasis through regulation of ISC proliferation and EC behavior in the Drosophila adult midgut. The sequential identification of the sSJ proteins Ssk, Mesh and Tsp2A has provided a Drosophila model system that can be used to elucidate the roles of the intestinal barrier function by experimental dysfunction of sSJs in the midgut. However, as described in this study, simple depletion of sSJ proteins throughout the adult midgut causes phenotypes that are too drastic, involving not only disruption of the intestinal barrier function but also intestinal dysplasia and subsequent lethality. To investigate the systemic effects of intestinal barrier impairment throughout the life course of Drosophila, more modest depletions of sSJ proteins are needed for future studies (Izumi, 2019).

Intestinal snakeskin limits microbial dysbiosis during aging and promotes longevity

Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. This study shows that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. These findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals (Salazar, 2018).

Aging is characterized by progressive health decline leading to mortality, yet the underlying pathophysiology remains elusive. There is an emerging understanding that maintaining intestinal barrier function during aging is critical to organismal health and longevity. At the same time, an age-related remodeling of epithelial junctions has been implicated in loss of barrier function in aged animals. These findings suggest that strategies to maintain epithelial junctions during aging may prove effective toward the goal of prolonging healthy lifespan. In this study, the fruit fly, Drosophila, was used as a model to study the impact of altered expression of the SJ-specific protein Ssk on intestinal barrier function, commensal homeostasis, and lifespan. Ssk was shown to be required to maintain barrier function and commensal homeostasis in the adult intestine. Indeed, loss of intestinal Ssk leads to rapid-onset microbial dysbiosis, immune gene modulation, barrier dysfunction, and mortality, although mortality was not due to dysbiosis. Furthermore, restoring Ssk led to resumption of barrier integrity and reversed these phenotypes, previously linked to aging. Remarkably, up-regulating Ssk in the adult intestine protected against oral infection with pathogenic bacteria. Improved survival under these conditions is linked to a reduction in bacterial translocation, consistent with improved intestinal barrier function. Finally, intestinal overexpression of Ssk during aging delayed the onset of dysbiosis and intestinal barrier dysfunction and prolonged lifespan. The prolongevity effects of Ssk were eliminated when flies were maintained axenically, consistent with a key role for Ssk-mediated alterations in microbiota dynamics for its effects on longevity (Salazar, 2018).

Previous work indicated that dysbiosis precedes and predicts intestinal barrier dysfunction in aged flies. This study shows that there is a mislocalization of SJs during midlife, before the detection of intestinal barrier failure. Hence, it is possible that alterations in SJs occur either before or concurrent with changes in microbial dynamics. It has previously been reported that age-related changes in SJ levels and localization were particularly noticeable at TCJs. Depletion of the Drosophila TCJ protein Gli led to ISC overproliferation and disruption of the intestinal barrier. Interestingly, however, acute loss of Gli does not lead to dysbiosis. This may be because TCJs contribute to only a small percentage of the barrier in the fly intestine, when compared with the bicellular junctions (Salazar, 2018).

Ssk forms a complex with Mesh, another sSJ-specific protein, and these proteins are mutually interdependent for their localization. Interestingly, it was recently reported that Mesh regulates Duox expression to modulate the load and composition of the gut microbiota. Consistent with this model, a significant inhibition of Duox expression was observed upon intestinal Ssk knockdown. At the same time, however, reduced expression was observed of several AMPs in Ssk knockdown flies. Furthermore, elevated AMP and Duox expression were observed after longer periods of Ssk knockdown, which is consistent with previously observed AMP elevations observed in Smurf flies and in aged non-Smurf flies. These data demonstrate that knocking down one SJ protein that disturbs intestinal junction integrity leads to an initial repression of AMPs that coincides with rapid elevations in microbial load, followed by an increase in immune activation, before death. These data provide a possible mechanism for the initial increase in microbial content observed before detectable barrier permeability, yet recapitulate the later elevation in immune gene expression observed in aged flies. Further studies are required to determine the mechanistic relationships between SJs, immune homeostasis, and microbiota dynamics during aging (Salazar, 2018).

In humans, compromised intestinal barrier function has been linked to a number of intestinal and systemic diseases. Although the existing clinical data have not established a causal role, the idea of targeting and restoring the intestinal epithelial barrier has been proposed as a potential therapeutic approach. A better understanding of the mechanisms underlying barrier regulation may aid this goal. At present, therapeutic approaches to treat intestinal barrier dysfunction have largely focused on reducing inflammation. Indeed, anti-tumor necrosis factor (TNF) therapy has been used to successfully treat intestinal barrier dysfunction in the context of Crohn disease. Consistent with this, TNF-deficient mice display improved intestinal barrier dysfunction during aging. Interestingly, anti-TNF therapy can reverse age-onset microbiota changes in mice. These data reveal that the composition of the microbiota can be altered by the inflammatory status of the host. Taken together, these studies support a model whereby an age-related increase in microbial translocation induces an inflammatory response, which contributes to dysbiosis. In this feedforward model, dysbiosis contributes to intestinal barrier dysfunction and increased microbial translocation. Previous work, in Drosophila, has shown that intestinal immune activation leads to intestinal barrier dysfunction and early-onset mortality. The current study adds to this model by showing that occluding junction modulation can, by itself, induce dysbiosis and mortality. In fact, under axenic conditions, and in the absence of an inflammatory response, occluding junction modulation, alone, is sufficient to induce certain markers of aging and mortality. This is consistent with an important role for SJs in the feedforward model. Given our findings, it will be important to determine whether interventions that maintain the intestinal epithelial barrier in aging mammals can prolong intestinal and/or organismal health during aging (Salazar, 2018).

Although Drosophila melanogaster is a well-studied and highly tractable model organism of proven utility in understanding mechanisms of aging, development, and disease, the relevance of molecular mechanisms uncovered in this work to human aging and disease remains to be demonstrated experimentally (Salazar, 2018).

The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, the c42-GAL4/UAS system was used to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport (Beyenbach, 2020).

The tetraspanins form a subset of the transmembrane 4 superfamily (TM4SF) of proteins that are highly conserved in eukaryotic organisms from plants to mammals. They are present in virtually every cell, and some cells have up to 100,000 copies of protein. Integral membrane proteins of small size (20–50 kDa), the tetraspanins first drew attention as 'clusters of differentiation' proteins (CD9, CD81, CD151, etc.) before they were found to share the sequence of tetraspanins. Protruding only for a short distance (3–5 nm) from membranes, tetraspanins are suited better for lateral than linear associations with each other and with other proteins. However, binding laterally to membrane proteins that extend into the extracellular space, tetraspanins may have enabled contact with other cells as the first step toward multicellular organisms. Today, tetraspanins are known to associate with integrins, cadherins, Rac, matrix-metalloproteases, EGF receptors and immunoglobulins, thereby serving scaffolding, cell adhesion, signal transduction, intracellular signaling, cell motility and migration, cell proliferation and differentiation, cell-cell fusion, endocytic trafficking, host-parasite interactions, metastasis, and viral infection (Beyenbach, 2020).

Tetraspanins are also found at paracellular junctions of epithelia. The tetraspanins Tspan3, CO-029, CD9, CD15, CD81, and CD181 associate with claudins in tight junctions of vertebrates. In invertebrates, tetraspanins are found in septate junctions. In particular, Tsp2A was found to localize to smooth septate junctions in the midgut and Malpighian tubules of Drosophila. The present study confirmed the location of Tsp2A in Malpighian tubules by localizing this protein in a well-defined band surrounding epithelial cells near the tubule lumen of Malpighian tubules of Drosophila. Sharing this band with Ssk and Mesh identifies Tsp2A as a protein of the smooth septate junction, as in Drosophila midgut epithelial cells. With length along the Malpighian tubule, the band of Tsp2A, Ssk, or Mesh forms a trapezoid ribbon dominated by principal cells that comprise 80% of tubule cells. The present study shows further that Dlg, the membrane-associated guanylate kinase (MAGUK) required for the formation of both septate and tricellular junctions and for epithelial polarization in Drosophila, is part of the trapezoid ribbon in Malpighian tubules. It suggests that Ssk, Mesh, Tsp2A, and Dlg form a complex at sSJs in Malpighian tubules and perhaps also in the midgut of Drosophila. Although Malpighian tubules and midgut utilize a single-layered epithelium for mediating transepithelial transport, they differ in the relative location of SJs and adherens junctions (AJs). In the midgut, sSJs are apical to AJs as in vertebrate tight junctions, while enigmatically the reverse is true for Malpighian tubules, even though Malpighian tubules and midgut share the same canonical polarization factors (Beyenbach, 2020).

The trapezoid ribbon of Tsp2A, Ssk, Mesh, and Dlg no longer forms upon the developmental knockdown of just one of these proteins, Tsp2A. The four septate junctional proteins remain largely in the cytoplasm of principal cells in close association, consistent with the need for the full complement of sSJ proteins before they can move to their usual paracellular location near the apical membrane. The few sSJs that can still be observed in Tsp2A knockdown tubules now spot the paracellular pathway for long distances with frequent gaps, wide gaps between the cell membranes of adjacent cells, and the loss of rungs in the ladder-like sSJ architecture. Junctions appearing at several places along the apical/basal axis indicate the diffuse distribution of sSJs along the paracellular pathway. Principal cells are also compromised. At basal surfaces, the basal lamina is unraveling consistent with the loss of structural support indicated by the increase in tubule diameter and hyperplasia. At the apical membrane, microvilli can be entirely absent; those present are shorter, less dense, and often devoid of mitochondria compared with control tubules. Thus the effects of knocking down Tsp2A extend beyond sSJs to the breakdown of the structural features of basolateral and apical plasma membranes in principal cells (Beyenbach, 2020).

The physiological consequences of the structural defects are severe. Malpighian tubules isolated from Tsp2A knockdown flies reared at 27°C failed to secrete fluid during their short life span less than a week. For one reason, apical microvilli, the sites that generate the driving force for transepithelial electrolyte secretion, are far less developed in Tsp2A knockdown tubules than in control tubules consistent with the diminished V-type H+-ATPase activity and the absence of significant lumen-positive transepithelial voltages in Tsp2A knockdown tubules. For another reason, the paracellular pathway has completely lost its barrier property in view of transepithelial input resistances that are not significantly different from the input resistances measured between the two measuring electrodes in Ringer solution. In the absence of paracellular barriers, the little solute and water still secreted into the tubule lumen via transcellular transport likely leak back to the peritubular bath (or hemolymph) via the paracellular pathway instead of flowing downstream for excretion. The tubular failure to excrete fluid is expected to disrupt extracellular fluid homeostasis as solute and water accumulate in the extracellular fluid compartment. The volume expansion leads to severe generalized edema and to death in the first week of adult life (Beyenbach, 2020).

In a recent study we found that Tsp2A, Ssk, and Dlg also remain largely in principal cells upon the developmental knockdown of mesh. Furthermore, mesh knockdown tubules fail to secrete fluid, and the flies gain extracellular fluid volume, become edematous, and die in early adulthood, as in the knockdown of Tsp2A. Similar effects of the knockdown of Tsp2A and mesh suggest that the full complement of septate junctional proteins consisting of Mesh, Tsp2A, Ssk, Dlg (and possibly other proteins) must be expressed before the sSJ can form in apicolateral regions in Malpighian tubules. The full complement of septate junctional proteins is indeed required for the formation sSJs in the midgut. There, the three proteins, Tsp2A, Mesh, and Ssk, are mutually dependent for their proper localization at the sSJ (Beyenbach, 2020).

The molecular mechanisms that bring about the structural and functional defects caused by the knockdown of Tsp2A were beyond the aims of the present study. Nevertheless, studies by others are relevant. Malpighian tubules are formed in the embryonic stage and become functional in the second half of the first day of development, responding in part to the 100-fold increase in uric acid by secreting this xanthine into the tubule lumen. From then on, the tubules do not undergo metamorphosis. Instead, they grow during the larval stages by increasing cell size associated with the endoreplication of DNA, and they acquire renal stem cells as late as the first day of pupation. Accordingly, the development of tumors and cysts upon the knockdown of Tsp2A must take place late in the development of the fly, which may explain why larvae survive the knockdown of Tsp2A or mesh (or other sSJ proteins) but not adults (Beyenbach, 2020).

Renal stem cells (RSCs) were first identified in Malpighian tubules as 'tiny cells' in the ureter and lower tubule of adult flies but not larvae. RSCs are thought to be multipotent, capable of generating all types of Malpighian tubule cells under autocrine regulation of JAK-STAT signaling, which stimulates RSCs to form renal blasts (RBs) that migrate and differentiate to stellate cells and principal cells in the upper tubule. Thus RSC activity can be expected to replace Tsp2A-deficient principal cells from the pupal stage on. Alas, the knockdown of Tsp2A may cause RSCs to produce Tsp2A-deficient principal cells, and failing to replace defective principal cells with normal cells, RSCs may remain active. Unstopped cell proliferation may then produce the hypertrophy, tumor- and cyst-like structures observed in the present study. Support for this hypothetical scenario in Malpighian tubules comes from the recent study of the role of sSJs in cell proliferation in the midgut of Drosophila. Here, enterocytes deficient of Tsp2A, Ssk, or Mesh exhibit defective sSJs, epithelial barrier dysfunction, abnormal enterocytes, and increased cell proliferation and intestinal hypertrophy, which the flies do not survive for more than 10 days. Thus the phenotype produced by knocking down Tsp2A in the intestine is similar to that of knocking down Tsp2A in Malpighian tubules. Moreover, the proliferation of intestinal stem cells (ISCs) was activated by Ras-MAP kinase and by the cytokines Unpaired2 (Upd2) and Unpaired3 (Upd3); the latter are known to activate JAK-STAT pathway involved in regulating RSC activity in Malpighian tubules. Similar molecular mechanisms in the intestine and Malpighian tubules suggest that septate junctional proteins and sSJs are critically involved in the regulation of stem cell proliferation in both tissues in as much as midgut intestinal stem cells (ISCs) arise from the same pool of midgut progenitor as RSCs in Malpighian tubules (Beyenbach, 2020).

Recent work has uncovered the critical role of Tsp2A in regulating stem cell proliferation in the Drosophila midgut via Hippo signaling. From fly to mammals, the Hippo pathway modulates cell proliferation, differentiation, and migration in developing tissues and limits growth in adults. First identified in Drosophila in 2003, the Hippo pathway has now grown to a signaling network of more than 40 players. One critical player is the transcription cofactor and oncogene Yorkie (Yki), which has been associated with Yki tumors and excessive body fluid retention described as 'bloating syndrome' in Drosophila. The Hippo pathway is regulated by components at or near cell junctions such as tight, septate, and adherens junctions. Transitions of components of the pathway [atypical protein kinase C (aPKC); Warts (Wts); and Hippo kinase (Hpo)] between the membrane and cytoplasm are thought to activate/inactivate the Hippo pathway while the shuttling of Yki between the cytoplasm and the nucleus is thought to regulate transcription and cell proliferation and apoptosis. Damage to the Drosophila midgut has been proposed to increase Yki activity and stimulate ISC proliferation. As the wound heals, Tsp2A undergoes internalization to facilitate the endocytic degradation of aPKC. Reduced aPKC activity allows Hpo to dimerize at the membrane, setting off a series of phosphorylations that ends with phosphorylated Yki and its cytoplasmic restriction, which stops ISC proliferation. Indeed, defects in Tsp2A expression or defective Tsp2A-sSJ assembly cause excessive aPKC-Yki-JAK-STAT activity and make the midgut epithelium highly proliferative, like a wound that cannot heal. Whether Hippo signaling is similarly activated in Tsp2A knockdown tubules will be of interest for future studies (Beyenbach, 2020).

A tetraspanin regulates septate junction formation in Drosophila midgut

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes via the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of SJs are observed: pleated SJs (pSJs) and smooth SJs (sSJs), which are present in ectodermally- and endodermally-derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Snakeskin (Ssk), in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh, identified a tetraspanin family protein, Tetraspanin 2A (Tsp2A), as a novel protein involved in sSJ formation in Drosophila. Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised (Tsp2A) expression caused by RNAi or the CRISPR/Cas9 system is associated with defects in the ultrastructure of sSJs, changes localization of other sSJ proteins, and impairs barrier function of the midgut. In most Tsp2A-mutant cells, Mesh fails to localize to sSJs and is distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, specialized intercellular junctions, designated as occluding junctions, regulate the free diffusion of solutes through the paracellular pathway. In vertebrates, tight junctions act as occluding junctions, whereas, in invertebrates, septate junctions (SJs) are the functional counterparts of tight junctions. SJs form circumferential belts around the apicolateral regions of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. SJs are subdivided into several morphological types that differ among different animal phyla, and several phyla possess multiple types of SJs that vary among different types of epithelia (Izumi, 2016).

In arthropods, two types of SJs exist: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectodermally-derived epithelia and surface glia surrounding the nerve cord, while sSJs are found mainly in endodermally-derived epithelia, such as the midgut and the gastric caeca. The outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs, although these epithelia are ectodermal derivatives. The criteria distinguishing these two types of SJs are the arrangement of the septa. In oblique sections of lanthanum-treated preparations, the septa of pSJs are visualized as regular undulating rows but those in sSJs are observed as regularly spaced parallel lines. In freeze-fracture images, the rows of intramembrane particles in pSJs are separated from one another, whereas those in sSJs are fused into ridges. To date, more than 20 pSJ-related proteins, including pSJ components and regulatory proteins involved in pSJ assembly, have been identified and characterized in Drosophila. In contrast, few genetic and molecular analyses have been carried out on sSJs. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, have been identified and characterized (Izumi, 2014; Izumi, 2012; Yanagihashi, 2012). Ssk consists of 162 amino acids and has four membrane-spanning domains, two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop (Yanagihashi, 2012). Mesh has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain, and a sushi domain (Izumi, 2012). Mesh transcripts are predicted to be translated into three isoforms of which the longest isoform consists of 1,454 amino acids. In Western blot studies, Mesh is detected as a main ~90 kDa band and a minor ~200 kDa band (Izumi, 2012). Compromised expression of ssk or mesh causes defects in the ultrastructure of sSJs and in the barrier function of the midgut against a 10-kDa fluorescent tracer (Izumi, 2012; Yanagihashi, 2012). Ssk and Mesh physically interact with each other and are mutually dependent for their sSJ localization (Izumi, 2012). Thus, Mesh and Ssk play crucial roles in the formation and barrier function of sSJs (Izumi, 2016).

Tetraspanins are a family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. Among several protein families with four transmembrane domains, tetraspanins are characterized especially by the structure of the second extracellular loop. It contains a highly conserved cysteine-cysteine-glycine (CCG) motif and 2 to 4 other cysteine residues. These cysteines form 2 or 3 disulfide bonds within the loop. Tetraspanins are believed to play a role in membrane compartmentalization and are involved in many biological processes, including cell migration, cell fusion and lymphocyte activation, as well as viral and parasitic infections. Several tetraspanins regulate cell-cell adhesion but none are known to be involved in the formation of epithelial occluding junctions. In the Drosophila genome, there are 37 tetraspanin family members, and some have been characterized by genetic analyses. Lbm, CG10106 and CG12143 participate in synapse formation. Sun associates with light-dependent retinal degeneration. TspanC8 subfamily members, including Tsp3A, Tsp86D and Tsp26D, are involved in the Notch-dependent developmental processes via the regulation of a transmembrane metalloprotease, ADAM10 (Dornier, 2012). However, the functions of most other Drosophila tetraspanins remain obscure (Izumi, 2016).

This study identified a tetraspanin family protein, Tsp2A, as a novel molecular component of sSJs in Drosophila. Tsp2A is required for sSJ formation and for the barrier function of Drosophila midgut. Tsp2A and two other sSJ-specific membrane proteins Mesh and Ssk show mutually dependent localizations at sSJs and form a complex with each other. Therefore, it is concluded that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

Of the sSJ-specific components, Mesh is a membrane-spanning protein and has an ability to induce cell-cell adhesion, implying that it is a cell adhesion molecule and may be one of the components of the electron-dense ladder-like structures in sSJs (Izumi, 2012). In contrast, both Ssk and Tsp2A are unlikely to act as cell adhesion molecules in sSJs because each of the two extracellular loops of Ssk (25 and 22 amino acids, respectively) appear to be too short to bridge the 15-20-nm intercellular space of sSJs. Furthermore, overexpression of EGFP-Tsp2A in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2016).

Several observations in Tsp2A-mutants may provide clues for understanding the role of Tsp2A in sSJ formation. In most Tsp2A-mutant midgut epithelial cells, Mesh fails to localize to the apicolateral membranes but was distributed in the cytoplasm, possibly to specific intracellular membrane compartments. To further examine where Mesh was localized in Tsp2A-mutant cells, the midgut was doublestained with the anti-Mesh antibody and the antibodies against typical markers of various intracellular membrane compartments, including the Golgi apparatus (anti-GM130), early endosomes (anti-Rab5), recycling endosomes (anti-Rab11) and lysosomes (anti-LAMP1). However, it was not possible to detect any overlap between staining by these markers and that of Mesh. The staining pattern in Tsp2A-mutant midgut epithelial cells produced with the anti-KDEL antibody, which labels endoplasmic reticulum, was similar, although not identical with that produced by the anti-Mesh antibody (Izumi, 2016).

Interestingly, some tetraspanins are known to control the intracellular trafficking of their partners. For instance, a mammalian tetraspanin, CD81 is necessary for normal trafficking or for surface membrane stability of a phosphoglycoprotein, CD19, in lymphoid B cells. The TspanC8 subgroup proteins, which all possess eight cysteine residues in their large extracellular domain, regulate the exit of a metalloproteinase, ADAM10, from the ER and differentially control its targeting to either late endosomes or to the plasma membrane (Dornier, 2012). Consequently, TspanC8 proteins regulate Notch signaling via the activation of ADAM10 in mammals, Drosophila and Caenorhabditis elegans. If Mesh is retained in the trafficking pathway from endoplasmic reticulum to plasma membrane in Tsp2A-mutant cells, Tsp2A may have an ability to promote the intracellular trafficking of Mesh in the secretory pathway. To clarify the role of Tsp2A in sSJ formation, it will be necessary to determine the intracellular membrane compartment where Mesh was localized in Tsp2A-mutant cells (Izumi, 2016).

Tsp2A, Mesh and Ssk are mutually dependent for their localization at sSJs. Consistent with this intimate relationship, the co-immunoprecipitation experiment revealed that Tsp2A physically interacts with Mesh and Ssk in vivo. However, the amount of Ssk observed in the co-immunoprecipitation with EGFP-Tsp2A was barely enriched relative to that in the extracts of embryos expressing EGFP-Tsp2A. This was particularly striking in comparison to the degree of enrichment of Mesh in the co-immunoprecipitation with EGFP-Tsp2A. To interpret these results, the detailed manner of the interaction between Tsp2A, Mesh and Ssk proteins needs to be further clarified. Many tetraspanin family proteins are known to interact with one another and with other integral membrane proteins to form a dynamic network of proteins in cellular membranes. Tetraspanins are also believed to have a role in membrane compartmentalization. Given such functional properties of tetraspanins, Tsp2A may determine the localization of sSJs at the apicolateral membrane region by membrane domain formation (Izumi, 2016).

In the Tsp2A-mutant midgut epithelial cells, Lgl was distributed throughout the basolateral membrane region, whereas it was localized in the apicolateral membrane region in the wild-type. In view of the role of Lgl in the formation of the apical-basal polarity of ectodermally-derived epithelial cells, it is of interest to consider whether this abnormal localization of Lgl in the Tsp2A-mutant affects epithelial polarity. However, in the Tsp2A-mutant midgut epithelial cells, Dlg still showed polarized concentration into the apicolateral membrane region and the Lgl never leaked into the apical membrane domain. These observations suggest that the lack of Tsp2A does not affect the gross apical-basal polarity of the midgut epithelial cells (Izumi, 2016).

Some tetraspanins have been reported to be involved in the regulation of cell-cell adhesion. A mammalian tetraspanin, CD151, regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin reorganization, or through complex formation with α3γ1 integrin. A mammalian tetraspanin, CD9, is concentrated in the axoglial paranodal region in the brain and in the peripheral nervous system, and CD9 knockout mice display defects in the formation of paranodal septate junctions and in the localization of paranodal proteins. Paranodal septate junctions have electron-dense ladder-like structures and their molecular organization is similar to that of pSJs but tetraspanins involved in pSJ formation have not been reported in Drosophila (Izumi, 2016).

Interactions between several tetraspanins and claudins, the key integral membrane proteins involved in the organization and function of tight junctions, are also known. Claudin-11 forms a complex with OAP-1/Tspan-3 and chemical crosslinking reveals a direct association between claudin-1 and CD9. Furthermore, the interaction between claudin-1 and CD81 is shown to be required for hepatitis C virus infectivity. To date, no tight junction defect has been reported in CD9 knockout mice, CD81 knockout mice, or CD9/CD81 double knockout mice. Further investigation is necessary to clarify whether the interactions between tetraspanins and tight junction proteins are involved in the formation and function of tight junctions (Izumi, 2016).

A novel protein complex, Mesh-Ssk, is required for septate junction formation in the Drosophila midgut

Septate junctions (SJs) are specialized intercellular junctions that restrict the free diffusion of solutes through the paracellular route in invertebrate epithelia. In arthropods, two morphologically different types of SJs have been reported: pleated SJs and smooth SJs (sSJs), which are found in ectodermally and endodermally derived epithelia, respectively. However, the molecular and functional differences between these SJ types have not been fully elucidated. This study reports that a novel sSJ-specific component, a single-pass transmembrane protein, which has been termed 'Mesh' (encoded by CG31004), is highly concentrated in Drosophila sSJs. Compromised mesh expression causes defects in the organization of sSJs, in the localizations of other sSJ proteins, and in the barrier function of the midgut. Ectopic expression of Mesh in cultured cells induces cell-cell adhesion. Mesh forms a complex with Ssk (Yanagihashi, 2012), another sSJ-specific protein, and these proteins are mutually interdependent for their localization. Thus, a novel protein complex comprising Mesh and Ssk has an important role in sSJ formation and in intestinal barrier function in Drosophila (Izumi, 2012).

Electron microscopic observations have shown that sSJs and pSJs can be distinguished morphologically. Obliquely sectioned pSJs and sSJs are visualized as regular undulating rows and regularly spaced parallel lines, respectively, while both types of SJs have ladder-like structures in the intermembrane space. Of the two sSJ-specific integral membrane proteins, Ssk is unlikely to be the structural element of the septa in sSJs, because its extracellular loops are both too short (25 and 22 a.a., respectively) to bridge the intercellular space. In contrast, Mesh induces cell-cell adhesion, implying that it may be one of the components of the septa observed in ultrathin section electron microscopy. Faint ladder-like structures were still observed in the mesh mutants, suggesting that other membrane proteins also contribute to the septal structures. FasIII is such a candidate because it shows cell-cell adhesion activity and was still distributed to the apicolateral region, as well as the apical region, in the mesh mutants. However, fasIII null mutant flies are viable and both Mesh and Ssk are normally localized at their sSJs, indicating that FasIII is dispensable for sSJ formation. FasIII may provide robustness to the Mesh-Ssk-mediated sSJ organization via its cell-cell adhesion activity (Izumi, 2012).

The issue of how SJs are organized in cells at the boundary between pSJ- and sSJ-bearing epithelia is intriguing. Interestingly, boundary cells were observed in which the pSJ marker Kune and sSJ marker Mesh were concentrated in the anterior and posterior regions, respectively, of the apicolateral membranes. This result suggests that individual cells possess both pSJs and sSJs depending on the orientation of their plasma membranes. The proventriculus is originally derived from ectoderm. However, the outer epithelial layer of the proventriculus (OELP) bears sSJs and expresses Mesh and Ssk, suggesting that the OELP has both ectodermal and endodermal characters. In fact, weak Kune expression was observed in the OELP but not in the midgut. Therefore, the boundary cell may have the ability to form either sSJs or pSJs according to the SJ type of adjacent cells. The occurrence of such 'SJ-boundary cells' seems to be crucial because they connect the ectodermally and endodermally derived epithelia into a tandem tube while maintaining the continuity of the paracellular barrier. However, the possibility that small amounts of pSJs and sSJs are also contained in the sSJs on the midgut side and pSJs on the foregut side of the SJ-boundary cells, respectively, to form hybrid junctions cannot be completely excluded (Izumi, 2012).

These analyses of Mesh and Ssk have clarified their interaction, interdependency in their localizations, and requirements for the organization and barrier function of sSJs, suggesting that Mesh-Ssk is a key system for sSJ formation. In mesh mutants, Ssk failed to localize at sSJs, but mislocalized to the apical and basolateral plasma membrane domains. In ssk-RNAi and Df(3L)ssk fly, Mesh no longer localized at the sSJs, but was distributed in the cytoplasm. Ssk may translocate Mesh from the cytoplasm to sSJs or to the plasma membrane. However, how the Mesh-Ssk complex recognizes and localizes to sSJ regions remain elusive. Mesh expression in S2 cells leads to cell aggregation without Ssk expression, suggesting that there is a mechanism by which Mesh translocates to the cell membrane and induces cell-cell adhesion independently of Ssk in S2 cells. Detailed analysis of the dynamics of Mesh-Ssk distribution will shed light on the mechanisms of sSJ formation and the sorting systems for sSJ proteins (Izumi, 2012).

By using Mesh and Ssk as specific markers for sSJs, it was confirmed that Dlg, Lgl and FasIII localize at sSJs in the larval OELP and midgut epithelial cells. In addition, it was found that Coracle (Cora) is also concentrated into sSJs. Among these proteins that are generally known as pSJ components, Lgl, Cora and FasIII were mislocalized in mesh mutants and ssk-RNAi lines. On the other hand, Lgl, Cora and FasIII were not required for the localization of Mesh and Ssk at the apicolateral membrane. These observations imply a possible hierarchy in the molecular constituents of sSJs; Mesh-Ssk might act as a platform for the assembly of Lgl, Cora and FasIII in endodermal epithelia. Such a feature in sSJs is in sharp contrast to that in pSJs where each molecular component is interdependent. Mutations in most of the genes encoding pSJ-associated proteins result in disruption of the barrier function and mislocalization of other pSJ proteins (Izumi, 2012).

Interestingly, in mesh mutants and ssk-RNAi lines, Dlg still localized at the apicolateral region of the OELP and midgut epithelial cells, although sSJs were disrupted at the ultrastructural level. Furthermore, Mesh and Ssk were distributed to the apicolateral region in dlg mutants, suggesting that Mesh-Ssk and Dlg are independent in their localizations. This is consistent with a recent report that Dlg is probably not a core pSJ component. Nevertheless, a functional relationship exists between Dlg and Lgl in determining cell polarity in ectodermally derived epithelia. Therefore, in the absence of Mesh and Ssk, Dlg may be unable to function properly because of an inadequate level of Lgl in the apicolateral regions. In fact, dlg^m52 and lgl⁴ maternal/zygotic mutants exhibited a similar hypertrophied midgut phenotype, suggesting that these proteins may function together in endodermal epithelia, as well as in ectodermal epithelia (Izumi, 2012).

The functions of Dlg, Lgl, Cora and FasIII at sSJs remain unknown. Dlg may act together with Lgl to regulate the apical-basal polarity in the early stage of epithelial development. In the late developmental stage, compensation mechanisms for the Dlg function may rescue the apicolateral localization of Mesh, as noted in ectodermally derived epithelial cells of dlg^m/z and lgl^m/z mutants. As larval midgut sSJs are completed at the end of embryogenesis (stage 17) in Drosophila, the organization of sSJ may not be influenced by early polarity defects of dlg^m/z and lgl^m/z mutants. Alternatively, Dlg and Lgl may be important for the regulation of the epithelial cell shape change that induces the midgut tube-like structure. In ectodermally derived epithelia, Coracle acts together with Yurt to regulate the apicobasal polarity. Thus, Cora and a Yurt-like molecule may function together to organize sSJs and/or to regulate the endodermal epithelial polarity (Izumi, 2012).

Homologous proteins, characterized by similar extracellular domains to Mesh, are present in vertebrates (e.g. mouse Susd2/SVS-1), implying that this family of proteins shares functions conserved across species. Mouse Susd2/SVS-1 has been suggested as a tumor-reversing gene product, because it inhibited the growth of cancer cell lines (Sugahara, 2007). Susd2/SVS-1 was distributed in the apical membrane of the epithelial cells in renal tubules and bronchial tubes, suggesting that it does not contribute to the cell-cell adhesion and/or paracellular barrier function in vertebrate epithelial cells. However, expressing Susd2/SVS-1 in HeLa cells induces the cell aggregation (Sugahara, 2007), implying that this protein family conserves the cell-cell adhesion activity. Further studies of the functions of Mesh-Susd2/SVS-1 family proteins in vertebrates and in invertebrates will lead to a better understanding of the conserved physiological functions in these proteins and of the evolution of intercellular junctions across species (Izumi, 2012).

Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila

Septate junctions (SJs) are the membrane specializations observed between epithelial cells in invertebrates. SJs play a crucial role in epithelial barrier function by restricting the free diffusion of solutes through the intercellular space. In arthropod species, two morphologically different types of SJs have been described: pleated septate junctions (pSJs) and smooth septate junctions (sSJs), which are specific to ectodermal and endodermal epithelia, respectively. In contrast to the recent identification of pSJ-related proteins, the molecular constituents of sSJs are mostly unknown. This study reports the discovery of a new sSJ-specific membrane protein, designated 'Snakeskin' (Ssk). Ssk is highly concentrated in sSJs in the Drosophila midgut and Malpighian tubules. Lack of Ssk expression is embryonically lethal in Drosophila and results in defective sSJ formation accompanied by abnormal morphology of midgut epithelial cells. It was also shown that the barrier function of the midgut to a fluorescent tracer is impaired in ssk-knockdown larvae. These results suggest that Ssk is required for the intestinal barrier function in Drosophila (Yanagihashi, 2012).

Search PubMed for articles about Drosophila Snakeskin

Beyenbach, K. W., Schone, F., Breitsprecher, L. F., Tiburcy, F., Furuse, M., Izumi, Y., Meyer, H., Jonusaite, S., Rodan, A. R. and Paululat, A. (2020). The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster. Am J Physiol Cell Physiol 318(6): C1107-C1122. PubMed ID: 32267718

Chen, H. J., Li, Q., Nirala, N. K. and Ip, Y. T. (2020). The Snakeskin-Mesh Complex of Smooth Septate Junction Restricts Yorkie to Regulate Intestinal Homeostasis in Drosophila. Stem Cell Reports 14(5): 828-844. PubMed ID: 32330445

Chen, J., Sayadian, A. C., Lowe, N., Lovegrove, H. E. and St Johnston, D. (2018). An alternative mode of epithelial polarity in the Drosophila midgut. PLoS Biol 16(10): e3000041. PubMed ID: 30339698

Dornier, E., Coumailleau, F., Ottavi, J. F., Moretti, J., Boucheix, C., Mauduit, P., Schweisguth, F. and Rubinstein, E. (2012). TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals. J Cell Biol 199: 481-496. PubMed ID: 23091066

Furuse, M. and Izumi, Y. (2017). Molecular dissection of smooth septate junctions: understanding their roles in arthropod physiology. Ann N Y Acad Sci 1397(1): 17-24. PubMed ID: 28636800

Izumi, Y., Yanagihashi, Y. and Furuse, M. (2012). A novel protein complex, Mesh-Ssk, is required for septate junction formation in the Drosophila midgut. J Cell Sci 125(Pt 20): 4923-4933. PubMed ID: 22854041

Izumi, Y., Motoishi, M., Furuse, K. and Furuse, M. (2016). A tetraspanin regulates septate junction formation in Drosophila midgut. J Cell Sci [Epub ahead of print]. PubMed ID: 26848177

Izumi, Y., Furuse, K. and Furuse, M. (2019). Septate junctions regulate gut homeostasis through regulation of stem cell proliferation and enterocyte behavior in Drosophila. J Cell Sci. 132(18). pii: jcs232108. PubMed ID: 31444286

Izumi, Y., Furuse, K. and Furuse, M. (2021). A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut. J Cell Sci. PubMed ID: 33589496

Jonusaite, S., Beyenbach, K. W., Meyer, H., Paululat, A., Izumi, Y., Furuse, M. and Rodan, A. R. (2020). The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule. Am J Physiol Cell Physiol 318(3): C675-C694. PubMed ID: 31913700

Resnik-Docampo, M., Koehler, C. L., Clark, R. I., Schinaman, J. M., Sauer, V., Wong, D. M., Lewis, S., D'Alterio, C., Walker, D. W. and Jones, D. L. (2017). Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis. Nat Cell Biol 19(1): 52-59. PubMed ID: 27992405

Salazar, A. M., Resnik-Docampo, M., Ulgherait, M., Clark, R. I., Shirasu-Hiza, M., Jones, D. L. and Walker, D. W. (2018). Intestinal snakeskin limits microbial dysbiosis during aging and promotes longevity. iScience 9: 229-243. PubMed ID: 30419503

Xu, C., Tang, H. W., Hung, R. J., Hu, Y., Ni, X., Housden, B. E. and Perrimon, N. (2019). The septate junction protein Tsp2A restricts intestinal stem cell activity via endocytic regulation of aPKC and Hippo signaling. Cell Rep 26(3): 670-688. PubMed ID: 30650359

Yanagihashi, Y., Usui, T., Izumi, Y., Yonemura, S., Sumida, M., Tsukita, S., Uemura, T. and Furuse, M. (2012). Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila. J Cell Sci 125(Pt 8): 1980-1990. PubMed ID: 22328496

date revised: 1 March 2024

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