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, 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.

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts.

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.

Molecular organization and function of invertebrate occluding junctions.

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.

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. 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.

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts.

Drosophila 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.

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes.

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.

The GC kinase Fray and Mo25 regulate Drosophila asymmetric divisions.

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 Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization.

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.

Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation.

The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence, ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together, our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.