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hoka: Biological Overview | References |

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, whereas 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 localization (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).

Search PubMed for articles about Drosophila Hoka or CG13704

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, 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

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 129(6): 1155-1164. 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). 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

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 e676. PubMed ID: 30650359

date revised: 5 August 2021

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