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Signaling pathways in Drosophila immunity
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drosophila relies on innate immunity to protect itself from its hostile environment throughout its life cycle. Despite the remarkable progress in understanding many aspects of Drosophila immunity, there are still big gaps in our knowledge. The general aim of this thesis was to gain a better understanding about the regulatory mechanisms controlling gene expression in Drosophila, with a focus on immunity. 

To enable isolation of Drosophila genes involved in immunity, we developed a method that allows visualization of immune gene expression in large number of embryos.  Reporter gene expression in wild type and mutant embryos was used to validate this approach, which should be a valuable complement to existing genetic and RNAi screens. 

Cactus, the Drosophila IκB protein, is known as a cytoplasmic inhibitor of Dif and Dorsal. We discovered that Cactus is also present in the cell nucleus. In response to Toll pathways signaling, cytoplasmic Cactus degrades rapidly in a proteasome-dependent manner, while a nuclear form of Cactus is stable and persists throughout signaling. This suggests that Cactus also has a function in the nucleus.

A genome-wide RNAi-based screen was performed in cultured S2 cells. Several novel components of NF-κB pathways were isolated as putative regulators of Drosophila immunity. One of them, the G protein-coupled receptor kinase-2 (Gprk2), was shown to be required for Drosomycin expression and for resistance to infection. Gprk2 interacts with Cactus, but is not required for Cactus degradation upon Toll pathway activation.  

The dpld/wech gene was previously found to affect periferal nervous system development. Here, we show that wech belongs to the LIN-41 subclade of the TRIM protein superfamily, and contains target sites for microRNAs. Genetic and cell transfection assays confirmed that wech expression is regulated by the microRNA let-7. This seems to be a conserved regulatory mechanism throughout the LIN-41 subclade. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University , 2011. , 67 p.
Keyword [en]
Drosophila immunity, signaling pathways, Cactus, NFκB
National Category
Natural Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-64256ISBN: 978-91-7447-398-8 (print)OAI: oai:DiVA.org:su-64256DiVA: diva2:456479
Public defence
2011-12-15, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2011-11-23 Created: 2011-11-14 Last updated: 2013-12-06Bibliographically approved
List of papers
1. Activation of an innate immune response in large numbers of permeabilized Drosophila embryos
Open this publication in new window or tab >>Activation of an innate immune response in large numbers of permeabilized Drosophila embryos
2011 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 35, no 3, 263-266 p.Article in journal (Refereed) Published
Abstract [en]

Innate immunity in Drosophila involves the inducible expression and synthesis of antimicrobial peptides. We have previously shown that not only Drosophila larvae and adults, but also embryos, are capable of mounting an immune response after injection of bacterial substances. To simplify genetic dissection of the signaling pathways involved in immune-gene regulation we developed a procedure for permeabilization of large number of embryos and subsequent infiltration with bacterial fragments. This approach, which promoted expression of CecropinA1- and Diptericin-driven β-gal expression in the epidermis of more than 90% of the treated embryos, will enable analysis of mutants that are embryonic lethal. Thus, genes that are involved in essential pleiotrophic functions, in addition to being candidates in immune-regulation will be amenable for analysis of their involvement in the fly's immune defense.

Keyword
Drosophila; Innate immunity, Antimicrobial peptide, NF-kappaB, Genetic screens, Insects
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-64253 (URN)10.1016/j.dci.2010.11.002 (DOI)000289871800005 ()21075135 (PubMedID)
Available from: 2011-11-14 Created: 2011-11-14 Last updated: 2017-12-08Bibliographically approved
2. Nuclear localization of the Drosophila IκB protein Cactus and its response to the Toll signaling pathway
Open this publication in new window or tab >>Nuclear localization of the Drosophila IκB protein Cactus and its response to the Toll signaling pathway
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In Drosophila, the Toll signaling pathway is known as a regulator of both dorso-ventral patterning during embryogenesis and regulation of immunity. Activation of of the Toll pathway results in nuclear accumulation of the NFκB/Rel transcription factors Dif and Dorsal, and the subsequent activation of downstream target genes. The current model is that Cactus is a strictly cytoplasmic protein, interacting with Dorsal and Dif to inhibit their nuclear translocation. However, immunostaining revealed that Cactus is present both in the nucleus and cytoplasm of fat body cells and S2 cells. Activation of Toll signaling in cell culture demonstrated that a nuclear form of Cactus is stable and persists during signaling, while cytoplasmic Cactus is degraded in a proteosome-dependent manner and then re-synthesized. Alternative splicing of Cactus pre-mRNA produces two Cactus isoforms, differing by 18 amino acids in the C-terminus. We show that both isoforms act as inhibotors of Dif- and Dorsal-mediated Drosomycin-luciferase expression, although the longer isoform of Cactus was a slightly better inhibitor. Both isoforms showed similar subcellular distribution, being present both in the cytoplasm and nucleus of larval fat body cells. Thus, the present finding suggest that Cactus does not act as a degradeable, cytoplasmic inhibitor of Dif and Dorsal, but also plays a role in the nucleus during immune challenge. 

National Category
Natural Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-64254 (URN)
Available from: 2011-11-14 Created: 2011-11-14 Last updated: 2011-11-15Bibliographically approved
3. Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-kappa B Signaling
Open this publication in new window or tab >>Genome-Wide RNA Interference in Drosophila Cells Identifies G Protein-Coupled Receptor Kinase 2 as a Conserved Regulator of NF-kappa B Signaling
Show others...
2010 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 184, no 11, 6188-6198 p.Article in journal (Refereed) Published
Abstract [en]

Because NF-kappa B signaling pathways are highly conserved in evolution, the fruit fly Drosophila melanogaster provides a good model to study these cascades. We carried out an RNA interference (RNAi)-based genome-wide in vitro reporter assay screen in Drosophila for components of NF-kappa B pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappa B regulators. Of these, nine dsRNA-treatments affect primarily the Toll pathway. G protein-coupled receptor kinase (Gprk) 2, CG15737/Toll pathway activation mediating protein, and u-shaped were required for normal Drosomycin response in vivo. Interaction studies revealed that Gprk2 interacts with the Drosophila I kappa B homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappa B regulation. Morpholino silencing of the zebrafish ortholog of Gprk2 in fish embryos caused impaired cytokine expression after Escherichia coli infection, indicating a conserved role in NF-kappa B signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappa B reporter activity. Gprk2 RNAi flies are susceptible to infection with Enterococcus faecalis and Gprk2 RNAi rescues Toll(10b)-induced blood cell activation in Drosophila larvae in vivo. We conclude that Gprk2/GRK5 has an evolutionarily conserved role in regulating NF-kappa B signaling. The Journal of Immunology, 2010, 184: 6188-6198.

National Category
Biological Sciences Microbiology in the medical area
Identifiers
urn:nbn:se:su:diva-49704 (URN)10.4049/jimmunol.1000261 (DOI)000278439600032 ()
External cooperation:
Note

authorCount :16

Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2017-12-11Bibliographically approved
4. Regulation of the Drosophila lin-41 Homologue dappled by let-7 Reveals Conservation of a Regulatory Mechanism Within the LIN-41 Subclade
Open this publication in new window or tab >>Regulation of the Drosophila lin-41 Homologue dappled by let-7 Reveals Conservation of a Regulatory Mechanism Within the LIN-41 Subclade
2008 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 237, no 1, 196-208 p.Article in journal (Refereed) Published
Abstract [en]

Drosophila Dappled (DPLD) is a member of the RBCC/TRIM superfamily, a protein family involved in numerous diverse processes such as developmental timing and asymmetric cell divisions. DPLD belongs to the LIN-41 subclade, several members of which are micro RNA (miRNA) regulated. We re-examined the LIN-41 subclade members and their relation to other RBCC/TRIMs and dpld paralogs, and identified a new Drosophila muscle specific RBCC/TRIM: Another B-Box Affiliate, ABBA. In silico predictions of candidate miRNA regulators of dpld identified let-7 as the strongest candidate. Overexpression of dpld led to abnormal eye development, indicating that strict regulation of dpld mRNA levels is crucial for normal eye development. This phenotype was sensitive to let-7 dosage, suggesting let-7 regulation of dpld in the eye disc. A cell-based assay verified let-7 miRNA down-regulation of dpld expression by means of its 3′-untranslated region. Thus, dpld seems also to be miRNA regulated, suggesting that miRNAs represent an ancient mechanism of LIN-41 regulation.

Keyword
dappled; Drosophila;let-7; LIN-41; miRNA; RBCC/TRIM
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-17729 (URN)10.1002/dvdy.21396 (DOI)000252386300019 ()
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2017-12-13Bibliographically approved

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