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Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3-5 ​kDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.

Organisms can generally be divided into two nutritional groups: generalists that consume various types of food and specialists that consume specific types of food. However, it remains unclear how specialists adapt to only limited nutritional conditions in nature. In this study, we addressed this question by focusing on Drosophila fruit flies. The generalist Drosophila melanogaster can consume a wide variety of foods that contain high glucose levels. In contrast, the specialist Drosophila sechellia consumes only the Indian mulberry, known as noni (Morinda citrifolia), which contains relatively little glucose. We showed that the lifespan of D. sechellia was significantly shortened under a high-glucose diet, but this effect was not observed for D. melanogaster. In D. sechellia, a high-glucose diet induced disorganization of the gut epithelia and visceral muscles, which was associated with abnormal digestion and constipation. RNA-sequencing analysis revealed that many immune-responsive genes were suppressed in the gut of D. sechellia fed a high-glucose diet compared with those fed a control diet. Consistent with this difference in the expression of immune-responsive genes, high glucose-induced phenotypes were restored by the addition of tetracycline or scopoletin, a major nutritional component of noni, each of which suppresses gut bacterial growth. We propose that, in D. sechellia, a high-glucose diet impairs gut immune function, which leads to a change in gut microbiota, disorganization of the gut epithelial structure and a shortened lifespan.

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, we used the c42-GAL4/UAS system 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.

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

Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh and Tsp2A. Here, we report a novel sSJ membrane protein, Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with 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 upregulated 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.

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal and renal system in arthropods. InDrosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. Here, we report a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region having 13-amino acids, and a cytoplasmic region with three repeats of the Tyr-Thr-Pro-Ala motif. The larval midgut inhoka-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 ofhokain the adult midgut leads to intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors. Inhoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion ofaPKCandykiinhoka-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 theDrosophilamidgut.Summary statementDepletion ofhoka, a gene encoding a novel septate junction protein, from theDrosophilamidgut results in the disruption of septate junctions, intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors.

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In

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, we used the c42-GAL4/UAS system 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.

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

Smooth septate junctions (sSJs) are cell-cell junctions that are thought to regulate the paracellular pathway of the intestine and renal system in arthropods. The detailed mechanism of action of sSJs is not well understood, because their molecular organization has remained elusive for a long time. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, were identified by screening monoclonal antibodies raised against sSJ-containing membrane fractions isolated from the silkworm midgut. Furthermore, a genetic screen in Drosophila based on microscopic observation of sSJ formation identified Tsp2A as a novel sSJ-specific membrane protein. Together with Tsp2A, Ssk and Mesh form a protein complex, and all three proteins are required for sSJ formation, as well as intestinal barrier function in Drosophila. Additional studies are likely to elucidate their roles in (1) the formation and reorganization of sSJs, (2) paracellular barrier functions and permselectivity, and (3) short-term and long-term regulation of paracellular functions in arthropod epithelia.

Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route of epithelial cells in theDrosophilamidgut. We previously 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. Here, we investigated the roles of sSJs in theDrosophilaadult 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, suppressed 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 theDrosophilaadult midgut.Summary statementDepletion of smooth septate junction-associated proteins from enterocytes in theDrosophilaadult midgut results in intestinal hypertrophy accompanied by accumulation of morphologically aberrant enterocytes and increased stem cell proliferation.

Septate junctions (SJs) are specialized intercellular junctions that function as permeability barriers to restrict the free diffusion of solutes through the paracellular routes in invertebrate epithelia. SJs are subdivided into several morphological types that vary among different animal phyla. In several phyla, different types of SJ have been described in different epithelia within an individual. Arthropods have two types of SJs: pleated SJs (pSJs) and smooth SJs (sSJs), found in ectodermally and endodermally derived epithelia, respectively. Several lines of Drosophila research have identified and characterized a large number of pSJ-associated proteins. Two sSJ-specific proteins have been recently reported. Molecular dissection of SJs in Drosophila and animals in other phyla will lead to a better understanding of the functional differences among SJ types and of evolutionary aspects of these permeability barriers.

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes through the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of septate junctions are observed; pleated (pSJs) and smooth (sSJs), which are present in ectodermally and endodermally derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and 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, Tsp2A, as a newly discovered 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 was associated with defects in the ultrastructure of sSJs, changed localization of other sSJ proteins, and impaired barrier function of the midgut. In most Tsp2A mutant cells, Mesh failed to localize to sSJs and was 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.

SummaryDrosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division.

Drosophila neuroblasts provide an excellent model for asymmetric cell divisions, where cell-fate determinants such as Miranda localize at the basal cortex and segregate to one daughter cell. Mechanisms underlying this process, however, remain elusive. We found that Mo25 and the GC kinase Fray act in this regulation. mo25 and fray mutants show an indistinguishable defect in Miranda localization. On the other hand, Drosophila Mo25 interacts with the tumor suppressor kinase Lkb1 in vivo, as have shown in mammals. Overexpression of Lkb1, which accumulates in the cell cortex, drastically relocalizes both Mo25 and Fray from the cytoplasm to the cortex, causing the same phenotype as mo25-mutant neuroblasts. Recovery from this defect caused by Lkb1 overexpression requires simultaneous overexpression of Mo25 and Fray. We suggest from those results that Mo25 and Fray operate together or in the same pathway in Drosophila asymmetric processes, and that their function counterbalances Lkb1.

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gbeta13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gbeta13F to the membrane, indicating an essential role of cortical Ggamma1-Gbeta13F signaling in asymmetric divisions. In Ggamma1 and Gbeta13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gbetagamma and other mutants indicates a predominant role of Partner of Inscuteable-Galphai in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.

The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCλ form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. In this paper, we report that an atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. Embryos depleted of PKC-3 by RNA interference die showing Par-like phenotypes including defects in early asymmetric divisions and mislocalized germline-specific granules (P granules). The defective phenotypes of PKC-3-depleted embryos are similar to those exhibited by mutants for par-3 and another par gene, par-6. Direct interaction of PKC-3 with PAR-3 is shown by in vitro binding analysis. This result is reinforced by the observation that PKC-3 and PAR-3 co-localize in vivo. Furthermore, PKC-3 and PAR-3 show mutual dependence on each other and on three of the other par genes for their localization. We conclude that PKC-3 plays an indispensable role in establishing embryonic polarity through interaction with PAR-3.

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. Here, we report 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. We also show 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.

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. Here, we report 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. We also show 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 .

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin-catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin-catenin system and may control cadherin-mediated cell-cell adhesion.

Follicular fluid (FF) and oocytes were obtained from 19 women for in vitro fertilization. Ovulation was induced with clomiphene citrate and human menopausal gonadotropin. Thirty-seven FF samples containing mature oocyte-corona-cumulus complexes were used to measure steroids, gonadotropins, and cAMP. The FF specimens were divided into three groups: follicles yielding ova that were fertilized and cleaved (group A), follicles containing fertilized ova without further cleavage (group B), and follicles containing nonfertilized oocytes (group C). The FF levels of progesterone, 17 beta-estradiol, LH/hCG, and FSH did not differ significantly among the groups. Mean FF cAMP concentrations declined from 17.5 +/- 2.5 pmol/mL 15 min after follicle aspiration to 3.6 +/- 0.9 pmol/mL after 3 h. The initial FF cAMP concentration did not differ significantly among three groups. However, the cAMP degradation rate in group A (119 +/- 13 X 10(-4) pmol/min) was significantly greater than that in group B (62 +/- 10 X 10(-4) pmol/min) or group C (75 +/- 8 X 10(-4) pmol/min). In conclusion, an increased intrafollicular cAMP degradation rate was associated with successful fertilization and cleavage of human oocytes in vitro. These data suggest that the degradation rate of FF cAMP may be a marker of optimal follicular development and oocyte maturation.

The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. Here, we identify Mushroom body defect (Mud) as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. We propose that Drosophila Mud, vertebrate NuMA and Caenorhabditis elegans Lin-5 (refs 5, 6) have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle.

When the apicolateral border of epithelial cells is compared with a polygon, its sides correspond to the apical junctional complex, where cell adhesion molecules assemble from the plasma membranes of two adjacent cells. On the other hand, its vertices correspond to tricellular contacts, where the corners of three cells meet. Vertebrate tricellular contacts have specialized structures of tight junctions, termed tricellular tight junctions (tTJs). tTJs were identified by electron microscopic observations more than 40 years ago, but have been largely forgotten in epithelial cell biology since then. The identification of tricellulin and angulin family proteins as tTJ-associated membrane proteins has enabled us to study tTJs in terms of not only the paracellular barrier function but also unknown characteristics of epithelial cell corners via molecular biological approaches.

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2–3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2–3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2–3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

UVC irradiation activates mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38. This study examined the role of protein kinase C (PKC) in the regulation of UVC-stimulated MAPKs activation. Either the depletion of PKC by prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or the inhibition of PKC by a selective PKC inhibitor, UCN-01-ME, attenuated UVC-activation of ERK1/2, keeping the activation of JNK1/2 intact. However, K252a, a non-selective PKC inhibitor, inhibited the activation of both ERK1/2 and JNK1/2 by UVC. In three isoforms of PKC (alpha, delta, epsilon) examined, PKC epsilon shows the most evident translocation, a temporal association with cell membrane, upon the UVC irradiation of NIH 3T3 cells. These results suggest that PKC is acting in the UVC-dependent activation of ERK1/2, and PKC epsilon is one of the PKC isozymes playing such a role.

West-Western screening of a cDNA expression library using 32P-labeled, autophosphorylated protein kinase Cdelta (PKCdelta) as a probe, led us to identify cDNA clones encoding a PKCdelta-binding protein that contains a leucine zipper-like motif in its N-terminal region and two PEST sequences in its C-terminal region. This protein shows overall sequence similarity (43.3%) to the serum deprivation response (sdr) gene product, and we named it SRBC (sdr-related gene product that binds to c-kinase). PKCdelta binds to the C-terminal half of SRBC through the regulatory domain and phosphorylates it in vitro. In COS1 cells, the phosphorylation of over-expressed SRBC is stimulated by 12-O-tetradecanoylphorbol-13-acetate and further enhanced by the over-expression of PKCdelta. The mRNA for SRBC is detected in a wide variety of cultured cell lines and tissues and is strongly induced by serum starvation. Furthermore, SRBC mRNA is induced during retinoic acid-induced differentiation of P19 cells. These results suggest that SRBC serves as a substrate and/or receptor for PKC and might be involved in the control of cell growth mediated by PKC.

Modulation of gene expression by 12-O-tetradecanoylphorbol-13-acetate (TPA) is thought to be mediated by protein kinase C (PKC), a major cellular receptor for TPA. We confirm this by showing that the overexpression of PKC delta enhances the TPA induction of the TRE-tk-CAT reporter gene in NIH3T3 cells. To investigate the mutual relationship between PKC delta- and Ras-dependent signal transduction pathways to a TRE binding transcription factor, AP1/Jun, we constructed constitutively active and dominant negative mutants of PKC delta. Activated Ras induced reporter gene expression in collaboration with overexpressed c-Jun or JunD, and this induction was insensitive to the dominant negative PKC delta. On the other hand, reporter gene expression induced by the constitutively active PKC delta was severely inhibited by dominant negative Ras, as well as by the dominant negative PKC delta. Thus, Ras activation must be indispensable for PKC delta to activate AP1/Jun. In the absence of overexpressed c-Jun or JunD, activated Ras was, however, clearly less effective than constitutively active PKC delta which showed full activation of reporter gene expression by itself. This suggests the presence of an additional, Ras-independent, signaling pathway downstream of PKC delta to activate AP1/Jun. In spite of the remarkable ability of constitutively active PKC delta to activate TRE-tk-CAT expression, this mutant suppressed cell growth.

第一胃フィスチュラをつけたホルスタイン種の成雌牛4頭を供試し,オーチャードグラス乾草,オーチャードグラスサイレージ,ビートパルプおよび濃厚飼料の給与が第一胃内揮発脂肪酸(VFA)の産生ならびに第一胃内性状に及ぼす影響を比較検討した.その結果次のことが認められた.1) 濃厚飼料の給与により,第一胃内pHは著しく低下した.ビートパルプ給与時のアンモニア態窒素濃度は極めて低く,他の飼料との間に有意差が得られた(P

乳熟後期(早刈)および黄熟後期(遅刈)刈取りのとうもろこしと出穂期(早刈)および開花後期(遅刈)刈取りのチモシー-ラジノクローバについて,それぞれサイレージを調製し,それらについて化学組成と発酵品質の分析を行い,去勢羊によって消化率と養分含量を求めた.またホルスタイン種の泌乳牛8頭を供試し,各サイレージの養分摂取量,乳量,乳組成についての測定,分析を行い,その産乳価値の差違を比較した.試験サイレージは自由に摂取させ,乾草は全牛に1日2kg,濃厚飼料は乳量の1/5を給与した.その結果,次のような知見を得た.1) サイレージ乾物摂取量および全TDN摂取量は,遅刈とうもろこし区が他の3区に比して,また早刈とうもろこし区は両牧草区に比して,それぞれ有意(P

第一胃フィステルを装着したホルスタイン種の成雌牛4頭を用い,とうもろこしサイレージの摂取量(1日当たり14,21,28,35kg)が第一胃内揮発性脂肪酸の産生ならびに第一胃内性状に及ぼす影響を検討した.その結果は次のとおりである.1) 摂取量により,第一胃内揮発性脂肪酸濃度が有意に変化した.また,各揮発性脂肪酸の割合において,酢酸に,摂取量による有意な変化が認められた.2) 摂取量の増加により,第一胃内揮発性脂肪酸濃度は直線的に上昇したが,第一胃内pHは低下した.また,各揮発性脂肪酸の割合において,酢酸は,摂取量の増加により直線的に減少したが,酪酸は増加した.プロピオン酸およびバレリアン酸には,摂取量による特定の傾向は認められなかった.

窒素施肥量(3-12kg/10a)が,チモシーを主体とする牧草サイレージの飼料価値に及ぼす影響を,2年間にわたって検討した.その結果,次のような知見を得た.1) 窒素多肥により,粗蛋白質の消化率およびDCP含量がそれぞれ有意(P

ホルスタイン種の泌乳牛8頭を供試し,とうもろこしサイレージ多給時におけるビートパルプおよび飼料用ビートの給与が,飼料摂取量,乳量および乳組成にどのような影響を及ぼすかを検討した.試験処理は,1) サイレージ,2) サイレージ+ビートパルプ,3) サイレージ+飼料用ビート,4) サイレージ+ビートパルプ+飼料用ビート,の4処理とした.とうもろこしサイレージ(完熟期刈取り)は自由給与とし,ビートパルプおよび飼料用ビートは乾物量で1日2.2kgを給与した.乾草は1日2kg,濃厚飼料は乳量の1/6を給与した.その結果次のような知見をえた.ビートパルプ,飼料用ビートおよびその両方の給与により,サイレージ乾物摂取量は,それらを併用しない場合よりも有意な減少を示したが,その減少量は,ビートパルプ,飼料用ビートおよびその両方の乾物摂取量とほぼ等しく,その結果,総乾物摂取量には各処理間にほとんど差がなく,またTDN, DCP摂取量にも各処理間に有意な差は認められなかった.ビートパルプ,飼料用ビートおよびその両方の給与により,乳量の増加する傾向が認められたが,いずれの処理間にも統計的有意差はえられなかった.また,乳組成においても各処理間に有意な違いは認められなかった.

ホルスタイン種の泌乳牛9頭を用い,サイレージ用とうもろこしの早生種である交4号(黄熟初期)および晩生種のジャイアンツ(乳熟初期)と1番刈りチモシー(出穂始期)について,それぞれサイレージを調製し,その産乳価値の差異を比較検討した.その結果,次のような知見をえた.1)サイレージの乾物摂取量において,ジャイアンッは交4号およびチモシーに比して有意(P

泌乳初期のとうもろこしサイレージの多給時において,濃厚飼料をFCM量の1/3と1/5給与した場合の飼料摂取量および産乳量に及ぼす影響の差異を明らかにするため,ホルスタイン種成雌牛10頭を供試し,分娩後14日目より14週間にわたって試験を実施した.黄熟後期刈取りのとうもろこしサイレージは自由に摂取させ,乾草は1日2kg給与した.その結果次のような知見を得た.濃厚飼料の多給により,サイレージの摂取量は減少したが,総乾物摂取量では増加する傾向が認められた.また,濃厚飼料の多給により,TDNおよびDCP摂取量がそれぞれ有意に増加した.全試験期間を通して,TDNおよびDCP摂取量は,濃厚飼料多給区においていずれも要求量を上回ったが,少給区では要求量を上回ったのはTDNのみで,DCPは全期間を通して要求量に達しなかった.乳量および乳組成には,濃厚飼料の給与最による有意な違いは認められなかった.にれらの結果から,十分登熟したとうもうもろこしサイレージを多給した場合,泌乳初期においても濃厚飼料の給与量を乳量の1/3以下に節減し得るものと考えられる.

第一胃フィステルを装着したホルスタイン種の成雌牛4頭を供試し,乾草,牧草サイレージおよびとうもろこしサイレージの各粗飼料と濃厚飼料の給与割合(100:0,75:25,50:50,25:75)が,第一胃内揮発性脂肪酸(VFA)の産生ならびに第一胃内性状に及ぼす影響の相違について比較検討した.その結果次のような知見を得た.1) 第一胃内pHは,粗飼料が乾草の場合,濃厚飼料の給与割合の増加に伴い直線的な低下を示した.2) 第一胃内アンモニア態室素濃度は,乾草の場合,濃厚飼料の増加により直線的な上昇を示したが,牧草サイレージの場合では,逆に減少を示した.3) 第一胃内VFA濃度は,両サイレージの場合,濃厚飼料の増加に反比例して減少した.4) 第一胃内各VFAの割合では,乾草の場合,濃厚飼料の増加とともに酢酸は減少し,酪酸およびイソバレリアン酸が増加した.一方,とうもろこしサイレージの場合,その増加とともに酪酸は減少を示したが,牧草サイレージの場合には,各VFAと濃厚飼料の給与割合との間に,特に一定の関係は認められなかった.

第一胃フィステルをつけたホルスタイン種の成雌牛4頭を用い,乾草の摂取量(1日当たり4,6,8,10kg)が第一胃内揮発性脂肪酸の産生ならびに第一胃内性状に及ぼす影響を検討した.その結果は次のとおりである.1) 摂取量により,第一胃内pH,アンモニア態窒素および揮発性脂肪酸濃度が有意に変化した.また,各揮発性脂肪酸の割合において,酢酸,酪酸およびiso-バレリアン酸に摂取量による有意な変化が認められたが,プロピオン酸およびn-バレリアン酸には変化がみられなかった.2) 摂取量の増加により,第一胃内pHは直線的に低下し,アンモニア態窒素および揮発性脂肪酸濃度は上昇した.3) 摂取量の増加により,各揮発性脂肪酸の割合において酢酸およびiso-バレリアン酸が直線的に低下し,酪酸は増加した.プロピオン酸およびn-バレリアン酸には,摂取量による特定の傾向は認められなかった.

第一胃フィステルを装着したホルスタイン種の成雌牛4頭を供試し,牧草サイレージの摂取量(1日あたり18,27,36,45kg)が第一胃内揮発性脂肪酸の産生ならびに第一胃内性状に及ぼす影響を検討した.その結果次のことが認められた.1)摂取量が異なることにより,第一胃内アンモニア態窒素および揮発性脂肪酸濃度が有意に変化した.また,各揮発性脂肪酸の割合において,酪酸に有意な変化が認められた.2)摂取量の増加により,第一胃内pHは直線的に低下し,アンモニア態窒素および揮発性脂肪酸濃度は上昇した.また,客揮発性脂肪酸の割合において,酢酸が直線的に低下し,酪酸は増加した.摂取量とプロピオン酸およびバレリアン酸との間には特定の関係は認められなかった.

第一胃フィステルを装着したホルスタイン種の成雌牛4頭を用い,乾草およびサイレージの給与が第一胃内揮発性脂肪酸(VFA)の産生ならびに第一胃内性状に及ぼす影響の差異を,刈取時期および窒素施用水準との関連で検討した.その結果次の知見を得た.1) 早刈,遅刈共通して,乾草に比べサイレージ給与時において第一胃内NH3-N濃度およびVFA中プロピオン酸,酪酸,バレリアン酸の割合が高く,酢酸の割合は低くなる傾向が認められた.2) 乾草,サイレージ共通して,早刈に比べ遅刈において第一胃内pHが高く,VFA濃度およびNH3-N濃度の低くなる傾向が認められた.3) 窒素施肥量の増加により,乾草,サイレージ共通して第一胃内NH3-N濃度が著しく高くなる傾向が認められた.