Daniel F. Woods

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.

REGULATION OF SYNAPSE STRUCTURE AND FUNCTION BY THE DROSOPHILA TUMOR SUPPRESSOR GENE DLG

Mutations of the tumor suppressor gene discs-large (dlg) lead to postsynaptic structural defects. Here, we report that mutations in dlg also result in larger synaptic currents at fly neuromuscular junctions. By selectively targeting DLG protein to either muscles or motorneurons using Gal-4 enhancer trap lines, we were able to rescue substantially the reduced postsynaptic structure in mutants. Rescue of the physiological defect was accomplished by presynaptic, but not postsynaptic targeting, consistent with our finding that miniature excitatory junctional currents were not changed in dlg mutants. These results suggest that DLG functions in the regulation of neurotransmitter release and postsynaptic structure. We propose that DLG is an integral part of a mechanism by which changes in both neurotransmitter release and synapse structure are accomplished during development and plasticity.

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.

The Partner of Inscuteable/Discs-Large Complex Is Required to Establish Planar Polarity during Asymmetric Cell Division in Drosophila

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

Localization of proteins to the apico-lateral junctions of Drosophila epithelia

We have examined the distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (DIg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. We have previously shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions [Woods et al., 1996]. We therefore extended this study to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and DIg antibodies, the apico-lateral junctional complexes appeared normal in tissue from the hyperplastic overgrowth mutants fat, dco, gd and wts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lgl showed abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signalling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signalling.

Isolation of cDNA clones encoding putative odourant binding proteins from the antennae of the malaria-transmitting mosquito, Anopheles gambiae.

One way of controlling disease transmission by blood‐feeding mosquitoes is to reduce the frequency of insect–host interaction, thus reducing the probability of parasite transmission and re‐infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub‐Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease‐transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.

Synaptic targeting and localization of Discs-large is a stepwise process controlled by different domains of the protein

BACKGROUND Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. Here, we have examined the targeting of the Drosophila MAGUK Discs-large (DLG) to larval neuromuscular junctions. RESULTS During development, DLG was first found associated with the muscle subcortical compartment and plasma membrane, and later was recruited to the postsynaptic membrane. Using a transgenic approach, we studied how mutations in various domains of the DLGprotein affect DLG targeting. Deletion of the HOOK region-the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain-prevented association of DLG with the subcortical network and rendered the protein largely diffuse. Loss of the first two PDZ domains led to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of DLG missing the GUK domain depended on the presence of endogenous DLG. CONCLUSIONS Postsynaptic targeting of DLG requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes DLG between compartments, possibly by interacting with recently identified cytoskeletal-binding partners.

Apical junctions and cell signalling in epithelia

SUMMARY Genetic analysis in Drosophila has led to the identification of several proteins that mediate cell-cell interactions controlling the fate and proliferation of epithelial cells. These proteins are localized or enriched in the adherens and septate junctions at the apical end of the lateral membranes between cells. The proteins localized or enriched at adherens junctions include Notch, which is important for the cell interactions controlling neuroblast and bristle patterning; Boss and sevenless, which are required for the cell interaction that establishes the R7 photoreceptor cell; and Armadillo, required for the wingless-dependent cell interactions that control segment polarity and imaginal disc patterning. Proteins localized at septate junctions include the product of the tumor suppressor gene dlg, which is required for septate junction formation, apical basal cell polarity, and the cell interactions that control proliferation. The results suggest that the cell signalling events important for cell fate determination and for cell proliferation control in epithelia occur at the apical junctions. The migration of the nucleus to the apical surface of the epithelium for mitosis may enable it to interact directly with the junction-associated signalling mechanisms.

Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains

MAGUKs (membrane-associated guanylate kinase homologs) are proteins involved in cell junction organization, tumor suppression, and signalling. Their structure includes one or three copies of a DHR or PDZ domain (discs-large homologous region or PSD-95/SAP90, discs-large ZO-1 homologous domain), an SH3 domain, and a guanylate kinase domain. MAGUKs were classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single DHR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides, camguk (cmg) is a Drosophila member of this novel MAGUK subfamily; we report its sequence and domain structure.