Sebzáródásban szerepet játszó gének azonosítása Drosophila melanogaster embrióban = Identification of genes involved in epithelial closure processes in Drosophila melanogaster

Beállitottunk és sikeresen alkalmaztunk egy nagyléptékű, RNS-interferencián alapuló, in vivo video mikroszkópiával kapcsolt funkcionális genomikai szűrést a Drosophila hámzáródási folyamataiban részt vevő gének azonosítására. Előzetes szűrésként génkifejeződési adatbázisokat vizsgáltunk meg és 6800 olyan gént táláltunk, melynek transzkripuma jelen van a fejlődő embrióban. Ezen géneknek megközelitőleg 40%-át, több mint 2500 gént, csendesitettük kettősszálú RNS-ek (dsRNS) mikroinjektálásával. Csaknem 100000 embriót kezeltünk dsRNS-sel, amiről 80000 filmet készitetünk és elemeztünk. Számos olyan új gént azonositottunk, amelyek szükségesek az embrionális háti záródáshoz. Az élő, dsRNS-sel kezelt embriókról készült filmek elemzése lehetővé tette a gécsendesitéssel előidézett fenotípus részletes leirását. Azonositottunk olyan géneket, melyek hiánya összehangolatlan záródást vagy a záródás teljes hiányát okozza. Jelenleg folytatjuk a funkcionális genomikai szűrést, illetve elkezdtük a szűréssel azonosított géneknek a torzáródásban betöltött szerepét vizsgálni. A sebzáródási esszé körülményeit vad tipusú embriókon beállitottuk és előkészitettük a dsRNS-sel kezelt embriók sebzáródási esszéit. | We have established and successfully applied an RNAi based genetic screen coupled with automated in vivo video microscopy to identify novel genes involved in epithelial closure processes. As a primary selection, we searched gene expression databases for genes whose transcripts are present in the embryo. This way, 6800 genes were found and selected for further analysis. DsRNAs for 2500 genes has been microinjected into ca. 100000 embryos, 80000 movies has been generated and analysed. Several novel genes has been shown to be involved in embryonic dorsal closure. Live imaging of the dsRNA-treated embryos enabled the identification and detailed phenotypic description of novel regulator genes of epithelial closure. Silencing of these genes caused abnormal dynamics of the closure process, misaligned epithelial sheets or complete lack of dorsal closure. Further analysis of the thorax closure of the adult flies will be an easy and quick method to confirm the function of our candidate genes in epithelial closure processes. Then, selected genes of which involvement in both the embryonic dorsal closure and thorax closure has been demonstrated will be subjected to wound healing assays. Parameters for the wound healing assays has been optimised and wound healing assays has been performed on wild type embryos

Kinetic studies of Candida parapsilosis phagocytosis by macrophages and detection of intracellular survival mechanisms

Peer reviewedPublisher PD

Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general

Nanobody-Displaying Flagellar Nanotubes

In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology

Characterization and functional analysis of zinc trafficking in the human fungal pathogen Candida parapsilosis

The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis-orthologues of ZRC1, ZRT3 and ZRT101-but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system

Cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in Drosophila melanogaster

Much of our knowledge on hematopoiesis, hematopoietic compartments, hematopoietic cell lineages and immunity has been derived from studies on the vertebrate immune system. The sophisticated innate immunity of insects, the phylogenetic conservation and the power of Drosophila genetics allowed the investigation of immune cell (hemocyte) lineage relationships in Drosophila melanogaster. The development of the hemocyte lineages in Drosophila is a result of a precisely regulated succession of intracellular and intercellular events, though the nature and extent of these interactions are not known. We describe here a cell lineage tracing system set up to analyze the development of hemocyte lineages and functionally distinct hemocyte subsets. This system allowed us to distinguish two major embryonic hemocyte lineages, the crq and Dot lineages, in two, physically separated compartments, the embryonic macrophages and the embryonic lymph gland. We followed the fate and development of these lineages in the construction of the larval hematopoietic compartments and during the cell-mediated immune response, the encapsulation reaction. Our results revealed the considerable plasticity and concerted action of the hematopoietic compartments and the hemocyte lineages in the development of the innate immune system and in the course of the cell-mediated immune response in Drosophila

Structural plasticity of the Salmonella FliS flagellar export chaperone

The Salmonella FliS flagellar export chaperone is a highly α-helical protein. Proteolytic experiments suggest that FliS has a compact core. However, the calorimetric melting profile of FliS does not show any melting transition in the 25-110 °C temperature range. CD measurements reveal that FliS is losing its helical structure over a broad temperature range upon heating. These observations indicate that FliS unfolds in a non-cooperative way and its native state shows features reminiscent of the molten globule state of proteins possessing substantial structural plasticity. As FliS has several binding partners within the cell, conformational adaptability seems to be an essential requirement to fulfill its multiple roles. This article is protected by copyright. All rights reserved

The spectraplakin short stop is an essential microtubule regulator involved in epithelial closure in Drosophila

Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. We investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium.In this study, we show that Shot is essential for the efficient final zippering of the opposing epithelial margins. Using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin-microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. FRAP analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule- binding activities of Shot are simultaneously required in the same molecule indicating that Shot is engaged as a physical crosslinker in this process.We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation which promotes zippering by initiating contacting of opposing epithelial cells