DRhoGEF2 Regulates Cellular Tension and Cell Pulsations in the Amnioserosa during Drosophila Dorsal Closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction

E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer

Human papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer

The Drosophila tricellular junction protein Gliotactin regulates its own mRNA levels through BMP-mediated induction of miR-184

Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at the Drosophila tricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3′UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184

The Drosophila tricellular junction protein Gliotactin regulates its own mRNA levels through BMP-mediated induction of miR-184

Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at the Drosophila tricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3′UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184

DRhoGEF2 and Diaphanous Regulate Contractile Force during Segmental Groove Morphogenesis in the Drosophila Embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis

Ageing results in loss of tissue homeostasis across taxa1. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability2. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases