Discs Large-1 Research Articles

Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells.

Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. Here, we uncover a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. We show that conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.

An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster

The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens.

PurposeWe previously showed that Discs large-1 (Dlg-1) regulates lens fiber cell structure and the fibroblast growth factor receptor (Fgfr) signaling pathway, a pathway required for fiber cell differentiation. Herein, we investigated the mechanism through which Dlg-1 regulates Fgfr signaling.MethodsImmunofluorescence was used to measure levels of Fgfr1, Fgfr2, and activated Fgfr signaling intermediates, pErk and pAkt, in control and Dlg-1–deficient lenses that were haplodeficient for Fgfr1 or Fgfr2. Immunoblotting was used to measure levels of N-cadherin, EphA2, β-catenin, and tyrosine-phosphorylated EphA2, Fgfr1, Fgfr2, and Fgfr3 in cytoskeletal-associated and cytosolic fractions of control and Dlg-1–deficient lenses. Complex formation between Dlg-1, N-cadherin, β-catenin, Fgfr1, Fgfr2, Fgfr3, and EphA2 was assessed by coimmunoprecipitation.ResultsLenses deficient for Dlg-1 and haplodeficient for Fgfr1 or Fgfr2 showed increased levels of Fgfr2 or Fgfr1, respectively. Levels of pErk and pAkt correlated with the level of Fgfr2. N-cadherin was reduced in the cytoskeletal-associated fraction and increased in the cytosolic fraction of Dlg-1–deficient lenses. Dlg-1 complexed with β-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3. EphA2 complexed with N-cadherin, β-catenin, Fgfr1, Fgfr2, and Fgfr3. Levels of these interactions were altered in Dlg-1–deficient lenses. Loss of Dlg-1 led to changes in Fgfr1, Fgfr2, Fgfr3, and EphA2 levels and to greater changes in the levels of their activation.ConclusionsDlg-1 complexes with and regulates the activities of EphA2, Fgfr1, Fgfr2, and Fgfr3. As EphA2 contains a Psd95/Dlg/ZO-1 (PDZ) binding motif, whereas Fgfrs do not, we propose that the PDZ protein, Dlg-1, modulates Fgfr signaling through regulation of EphA2.

Abstract 4408: Her2 C terminal PDZ binding domain interacting with DLG1 is required for radioresistance through induction of EMT and loss of epithelial polarity

Abstract The tyrosine kinase receptor HER2 (ErbB2/neu) is predominantly localized to the basolateral membrane of the epithelium and has been implicated in mammary tumorigenesis as well as in resistance to chemotherapy and radiation. Here we demonstrate that HER2 interacts with discs large 1 (DLG1) that is one of PDZ domain proteins expressed at the basolateral membrane of human breast epithelial cells and plays an important role in the establishment and maintenance of epithelial polarity in Drosophila. However, either loss of the last 6 C-terminal amino acids (HER2-Δ1250-1255) or a valine-to-alanine C-terminal substitution (HER2-V1255A) in HER2, abrogates the interaction with DLG1. In contrast, either PDZ domain 1 or 3 within Dlg1 is required for interaction with HER2. Moreover, HER2-Δ1250-1255- or HER2-V1255A-expressing MCF10A human breast epithelial cells show the reduction of mesenchymal-to-epithelial transition (EMT) proteins including vimentin and Snail. In addition, HER2-wt cells promotes EMT as shown by down-regulation of E-cadherin and upregulation of vimentin and Snail. Taken together, these results indicate that C-terminal PDZ binding domain in HER2 modulates biological functions of HER2. These results reveal new insight into the structure and function of HER2, which may guide the design and development of new anti-cancer targets to treat HER2-positive breast cancer. Citation Format: Dong Lin, Xiaodi Zhang, Angela Eldridge, Ming Fan, Cheikh Menaa, Jianjian Li. Her2 C terminal PDZ binding domain interacting with DLG1 is required for radioresistance through induction of EMT and loss of epithelial polarity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4408. doi:10.1158/1538-7445.AM2014-4408

DLG1 influences distal ureter maturation via a non-epithelial cell autonomous mechanism involving reduced retinoic acid signaling, Ret expression, and apoptosis

The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used RetGFP knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.

Novel mechanism of tumor suppression by polarity gene discs large 1 (DLG1) revealed in a murine model of pediatric B-ALL.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel "pre-leukemic" stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers.

N-glycosylation requirements in neuromuscular synaptogenesis

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:α-3-D-mannoside-β1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)GL] scaffolds. We conclude that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis.

The internal structure of embryonic gonads and testis development in Drosophila melanogaster requires scrib, lgl and dlg activity in the soma

Interest in the mechanism leading to the formation of the germline and its differentiation during Drosophila development, initiated even as soon as the first ever cloned tumour suppressor gene in Drosophila, the lethal (2) giant larvae (lgl), had been identified. Further work has shown that the lgl, as well as discs large-1 (dlg) and scribble (scrib) tumor suppressor genes code for scaffolding proteins associated with either the cytoskeletal matrix or the septate junctions that act in common pathways in various tissues. This study analysed the role of Dlg, Scrib and Lgl in the embryonic gonads and testis of Drosophila melanogaster. Loss of scrib, dlg and lgl had no effect on gonad formation, but Dlg and Scrib in the gonadal mesoderm acted critically in the somatic wrapping of the pole cells and the internal structure of the Drosophila embryonic gonads. Dlg also affected the incorporation of the male-specific Sox100B positive mesodermal cells into the male embryonic gonads, yet Sox100B expression in dlg testis remained unaffected. Analysis at later stages revealed that scrib and lgl expression in the somatic lineage of the Drosophila testis, similar to what was previously shown for dlg, was indispensable for testis development and homeostasis, as depletion of these genes resulted in extensive testes defects. The data presented here emphasize the somatic requirement of Scrib, Dlg and Lgl in embryonic gonads, as well as in the Drosophila testis that underlines the importance of the somatic lineage in the establishment and maintenance of testis formation throughout successive developmental stages.

Molecular Basis for the Recognition of Adenomatous Polyposis Coli by the Discs Large 1 Protein

The human Discs Large 1 (DLG1) protein uses two of its three PDZ domains to interact with the C-terminal peptide of the Adenomatous Polyposis Coli (APC) tumor suppressor protein. The DLG1/APC complex inhibits the cell cycle progression from the G0/G1 to the S phase, regulates epithelial cell migration and morphogenesis, and is required for polarization of the microtubule cytoskeleton. However, the molecular details of how DLG1 recognizes APC is not clear. In this study, we performed biochemical and biophysical assays to investigate the interactions between PDZ domains of DLG1 and the C-terminal peptide of APC. In addition, we determined the crystal structures of the PDZ1 and PDZ2 domains of DLG1 each in complex with the C-terminal 11-residue peptide of APC. Our biochemical, biophysical, and structural results revealed structural elements and residues on PDZ1 and PDZ2 domains of DLG1 and on APC crucial for their mutual interaction. In particular, our results show that the β2/β3 loops of PDZ1 and PDZ2 play important roles in contributing to the binding affinities between PDZ domains and APC, through interacting with the residues upstream of the canonical PDZ-binding S/T-X-V motif. The results provide new insights into the binding mode of a defined C-terminal segment of APC by the PDZ domains of DLG1.

Involvement of protein kinase Cα in invadopodia formation in Src‐3T3 cells

Dynamic structures termed podosomes or invadopodia take the form of actin puncta or rings and are involved in extracellular matrix remodeling, adhesion, and invasion. Though various protein kinase C (PKC) isoforms are known to affect these structures, the precise role of PKCα in signaling upstream of invadopodia in transformed cells remains unclear. We have begun to analyze the role of PKCα in NIH 3T3 fibroblasts transformed with active Src (Src‐3T3 cells). PKCα colocalizes with actin and the invadopodia marker Tks5 at rosettes, and inhibition of conventional PKC isoforms interferes with PKCα localization and rosette formation. The target of PKC that is responsible for invadopodia formation is unknown; however, one possible candidate is Discs Large 1 (DLG1), which we show is a substrate of PKC. DLG1 is present at numerous actin‐based structures including invadopodia and is involved in motility and invasion. Strikingly, coexpression of DLG1 and PKCα in COS‐7 epithelial cells causes formation of actin‐based rosettes (in a cPKC‐dependent manner), suggesting that phosphorylated DLG is sufficient for establishment of these structures.This work was supported by NIH P01 DK54441 and NIH GM43154.

Characterization of a Novel Human Cell-Cycle-Regulated Homologue of Drosophila dlg1

Cell cycle defects have been associated with the process of carcinogenesis in many studies. Here we report the cloning and analysis of the novel gene KIAA0008 (GenBank acc. no. D13633). Chromosomal localization experiments assigned the gene to chromosome 14q22–q23. The mRNA transcript was found to be cell cycle regulated, expressed at S-phase, and maintained at both G2-and M-phases. In situ hybridization showed expression in proliferating colon and breast (tumor) tissues. Structurally, KIAA0008 shares homology with the Drosophila melanogaster discs large-1 (dlg1) tumor suppressor gene and membrane-associated guanylate kinase protein family members. The potential involvement of KIAA0008 in cell proliferation is discussed, along with its sequence identity and tissue distribution.

Isolation and Characterization of a Mouse Homolog of the Drosophila Segment Polarity Gene dishevelled

In the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2) . Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.

The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.

Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs. Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins. Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction. The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia.

Molecular cloning of the lethal(1)discs large-1 oncogene of Drosophila

We present a genetic, developmental and molecular analysis of lethal(1)discs large-1 [ l(1)d.lg-1; Stewart et al., 1972], an oncogene of Drosophila. Mutations in this gene cause the imaginal discs to grow by cell proliferation beyond their normal final size, transform into solid tumors, fuse with one another and the brain, and lose their ability to differentiate. The oncogene represents the only known complementation group between two deficiency breakpoints, and 15 recessive lethal alleles are available. Cloning of the DNA between the two deficiency breakpoints defines a region of 45 ± 2 kb. The l(1)d.lg-1 transcription unit is identified by both qualitative and quantitative effects of several l(1)d.lg-1 mutations on the RNA transcripts and by the presence of a DNA insert in one of the l(1)d.lg-1 alleles. It gives rise to at least five different transcripts ranging in size from 1.9 to 6.0 kb. Three other transcription units are present within this region, two 5′ to the l(1)d.lg-1 gene and one at the 3′ end. A near full-length cDNA from one of the larger transcripts of l(1)d.lg-1 has homology to genomic DNA spanning over 20 kb. A developmental profile of l(1)d.lg-1 transcription is presented. We discuss how mutations in this gene could disrupt epithelial structure and how this might be related to the excessive cell proliferation and interdisc fusion that is observed. We also compare this gene with another recessive oncogene of Drosophila, lethal(2)giant larvae, that has been cloned and characterized.