[Show abstract][Hide abstract] ABSTRACT: Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.
Preview · Article · Apr 2010 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.
No preview · Article · Apr 2008 · European Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.
Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.
Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.
Full-text · Article · Feb 2008 · BMC Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: The development of a technique to stably integrate exogenous DNA into the germline of Drosophila melanogaster marked a milestone in the ability to study gene function in the fly. On the molecular level germline transformation mainly relies on a particular transposable element, the D. melanogaster P-element. Based on certain features of the P-element, vectors have been designed for diverse applications like gene disruption, chromosome engineering, gene tagging, and inducible gene expression/repression. Despite the fact that an increasing number of other transposons have been utilized for germline transformation of Drosophila most transformation vectors are still P-element based. Technically, microinjection serves as the method of choice to physically introduce transgenes into preblastoderm Drosophila embryos. Besides an appropriate technical equipment including suitable microcapillaries in conjunction with a micromanipulator, a microinjector, and a microscope, proper handling of the Drosophila embryos before and after microinjection is the key step to the generation of transgenic flies. Pioneer work in Drosophila also served as a general guideline for the transformation of other insect species including those with medical and agricultural importance.
Preview · Article · Jan 2008 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The formation and maintenance of cilia and flagella require a selective and directed transport along the axoneme, a characteristic central bundle of microtubules. Recent evidence suggests an interesting link between the generation of cilia and the protein complexes that establish apico-basal cell polarity.
[Show abstract][Hide abstract] ABSTRACT: Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, we analyzed the subcellular distribution of Drosophila Lin-7 (DLin-7) and performed genetic and biochemical assays to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses.
Full-text · Article · May 2004 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.
[Show abstract][Hide abstract] ABSTRACT: The Drosophila gene Serrate encodes a membrane spanning protein, which is expressed in a complex pattern during embryogenesis and larval stages. Loss of Serrate function leads to larval lethality, which is associated with several morphogenetic defects, including the failure to develop wings and halteres. Serrate has been suggested to act as a short-range signal during wing development. It is required for the induction of the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. In order to understand the regulatory network required to control the spatially and temporally dynamic expression of Serrate, we analysed its cis-regulatory elements by fusing various genomic fragments upstream of the reporter gene lacZ. Enhancer elements reflecting the expression pattern of endogenous Serrate in embryonic and postembryonic tissues could be confined to 26 kb of genomic DNA, including 9 kb of transcribed region. Expression in some embryonic tissues is under the control of multiple enhancers located in the 5' region and in intron sequences. The data presented here provide the tools to unravel the genetic network which regulates Serrate during different developmental stages in diverse tissues.
No preview · Article · Sep 1998 · Development Genes and Evolution
[Show abstract][Hide abstract] ABSTRACT: The product of the Drosophila gene Serrate acts as a short-range signal during wing development to induce the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the dorsal compartment of the wing disc have recently been confined to a genomic fragment in the 5'-upstream region of the gene. Here we present data to suggest that this fragment responds to various positive and negative inputs required for the early Serrate expression. First, activation and maintenance of expression in the dorsal compartment of the wing discs of second and early third instar larvae depends on apterous, as revealed by reporter gene expression in discs either lacking or ectopically expressing apterous. Second, transcriptional downregulation during third larval instar is mediated by hiiragi. Finally, this regulatory element responds to Delta signalling in a nonautonomous way to maintain Serrate expression along the dorsal margin. The results clearly show that some of the previously described transactivators of Serrate protein expression, e.g. fringe, act on elements required for later aspects of Serrate expression.
Preview · Article · Aug 1998 · Mechanisms of Development