Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.     

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.     

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.     

discs large in the Drosophila testis

Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division, and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.     

The claudin-like megatrachea is essential in septate junctions for the epithelial barrier function in Drosophila

Vertebrate claudin proteins are integral components of tight junctions, which function as paracellular diffusion barriers in epithelia. We identified Megatrachea (Mega), a Drosophila transmembrane protein homologous to claudins, and show that it acts in septate junctions, the corresponding structure of invertebrates. Our analysis revealed that Mega has transepithelial barrier function similar to the claudins. Also, Mega is necessary for normal tracheal cell morphogenesis but not for apicobasal polarity or epithelial integrity. In addition, we present evidence that Mega is essential for localization of the septate junction protein complex Coracle/Neurexin. The results indicate that claudin-like proteins are functionally conserved between vertebrates and Drosophila.     

Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160- kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase- like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs- large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double- label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.     

The Drosophila cell survival gene discs lost encodes a cytoplasmic Codanin-1-like protein, not a homolog of tight junction PDZ protein Patj

The Drosophila gene discs lost (dlt) has been reported to encode a homolog of the vertebrate tight junction PDZ protein Patj, and was thought to play a role in cell polarity. Using rescue experiments and sequence analyses, we show that dlt mutations disrupt the Drosophila Codanin-1 homolog, a cytoplasmic protein, and not the PDZ protein. Mutations in human Codanin-1 are associated with congenital dyserythropoietic anemia type I (CDA I). In Drosophila, the genomic organization of dlt is unusual. dlt shares its first untranslated exon with alpha-spectrin, and both genes are coexpressed throughout development. We show that dlt is not required for cell polarity but is needed for cell survival and cell cycle progression. Finally, we present evidence that the PDZ protein previously thought to be encoded by dlt is not required for viability. We propose to rename this PDZ protein after its vertebrate homolog, Patj (Pals-associated tight junction protein).     

DLG1: Chromosome Location of the Closest Human Homologue of the Drosophila Discs Large Tumor Suppressor Gene

The Drosophila discs large tumor suppressor protein, Dlg, is the prototype of a newly discovered family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). MAGUKs are localized at the membrane-cytoskeleton interface, usually at cell-cell junctions, where they appear to have both structural and signaling roles. They contain several distinct domains, including a modified guanylate kinase domain, an SH3 motif, and one or three copies of the DHR (GLGF/PDZ) domain. Recessive lethal mutations in the discs large tumor suppressor gene interfere with the formation of septate junctions (thought to be the arthropod equivalent of tight junctions) between epithelial cells, and they cause neoplastic overgrowth of imaginal discs, suggesting a role for cell junctions in proliferation control. A homologue of the Dlg protein, named Hdlg, has been isolated from human B lymphocytes. It shows 65-79% identity to Dlg in the different domains, and it binds to the cytoskeletal protein 4.1. Here, we report that the gene for lymphocyte Hdlg, named DLG1, is located at chromosome band 3q29. This finding identifies a novel site for a candidate tumor suppressor on chromosome 3.     

Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila

Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithelial junction assembly and morphogenesis.     

The Drosophila selenophosphate synthetase (selD) gene is required for development and cell proliferation

To study the function of selenoproteins in development and growth we have used a lethal mutation (selD(ptuf)) of the Drosophila homologous selenophosphate synthetase (selD) gene. This enzyme is involved in the selenoprotein biosynthesis. The selD(ptuf) loss-of-function mutation causes aberrant cell proliferation and differentiation patterns in the brain and imaginal discs, as deduced from genetic mosaics, patterns of gene expression and analysis of cell cycle markers. In addition to that, selenium metabolism is also necessary for the ras/MAPKinase signal tansduction pathway. Therefore, the use of Drosophila imaginal discs and brain and in particular the selD(ptuf) mutation, provide an excellent model to investigate the role of selenoproteins in the regulation of cell proliferation, growth and differentiation.     

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co-overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway.     

Drosophila S2 Cells Secrete Wingless on Exosome‐Like Vesicles but the Wingless Gradient Forms Independently of Exosomes

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless‐expressing Drosophila S2 cells, Wingless is present on exosome‐like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless‐containing exosome‐like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome‐like vesicles. Using these exosome markers and a cell‐based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless‐expressing cells.     

rDLG6: a novel homolog of Drosophila DLG expressed in rat brain

Drosophila DLG (Discs Large Tumor Suppresser Protein) is a component of septate junctions, and disruption of its gene leads to over growth of imaginal discs. Homologs of Drosophila DLG recently isolated from mammalian tissue have been classified as members of the MAGUK (Membrane Associated GUanylate Kinase) superfamily of proteins. Using a modified RT-PCR method applied to rat tissues, we have isolated cDNA clones encoding a novel MAGUK family member that we have named rDLG6. Immunoblot and immunohistochemical analyses revealed that rDLG6 protein is predominantly expressed in brain. GST pull-down assays showed that the PDZ domain of rDLG6 protein binds to the C-terminus of the AMPA (alpha-Amino-3-hydroxy-5-Methyl-isoxazole-4-Propionic Acid) receptor GluR2 subunit.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies on tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations resulted in the formation of blisters in the wings, which was due to a defective wing unfolding process. Interestingly, by(1)-the mutant allele of the gene blistery (by)-also showed a blistered wing phenotype, but failed to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, we have demonstrated that tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increased JNK activity and induced apoptotic cell death. Collectively, our data suggest that tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development.