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Genome-wide RNA interference in Drosophila cells identifies G protein-coupled receptor kinase 2 as a conserved regulator of NF-κB signaling
University of Tampere. (Hultmark)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Hultmark)
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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-kappaB 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-kappaB pathways. We analyzed 16,025 dsRNA-treatments and identified 10 novel NF-kappaB 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 IkappaB homolog Cactus, but is not required in Cactus degradation, indicating a novel mechanism for NF-kappaB 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-kappaB signaling. Moreover, small interfering RNA silencing of the human ortholog GRK5 in HeLa cells impaired NF-kappaB 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-kappaB signaling.

Place, publisher, year, edition, pages
American association of immunologists , 2010. Vol. 184, no 11, 6188-6198 p.
National Category
Microbiology Cell and Molecular Biology
URN: urn:nbn:se:umu:diva-38693DOI: 10.4049/jimmunol.1000261ISI: 000278439600032PubMedID: 20421637OAI: diva2:380608
Available from: 2010-12-21 Created: 2010-12-21 Last updated: 2014-09-08Bibliographically approved
In thesis
1. Toll-mediated cellular immune response in Drosophila melanogaster
Open this publication in new window or tab >>Toll-mediated cellular immune response in Drosophila melanogaster
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects are amongst the most abundant and diversified multi-cellular organisms on earth. As pollinators of the vast majority of our food crops their socio-economic value is hard to overestimate. Although many pest and pathogens of the honeybee have been known for decades, we still fail to explain the huge losses of honeybee colonies in recent years.At the beginning of my PhD studies, I investigated the effect that senescence and the age-related caste dimorphisms have on two basic parameters of the adult honeybee’s immune system, namely blood cell concentration and the activity of the phenoloxidase cascade. Realizing the limitations of working on an organism for which (at the time) no sequenced genome or molecular tools were available, I switched labs to work on Drosophila melanogaster. The fruit fly has proven to be a particularly useful model system to identify and study genes critical for both the innate immune response itself, as well as the signaling pathways regulating it. For the main part of my thesis, I used the tissue-specific expression of fluorescent markers to visualize segmentally aligned bands of sessile blood cells in the Drosophila larva. This phenotype is disturbed in larvae heterozygote for a gain-of-function mutation in the Toll pathway called Tl10b. In a genetic screen, I scored the ability of genomic mutations to modify the Tl10b loss of bands phenotype. I identified five genomic regions that suppressed the disturbed band pattern of sessile blood cells, and in three of these regions I mapped down this phenotype to single gene level. Two genes are involved in intracellular vesicle trafficking (Rab23 and ird1) and one is activated at the onset of metamorphosis (hdc). To confirm the experimental model, I tested the role of another negative regulator of the Toll pathway. I used tissue specific GAL4 fly lines to express RNAi silencing constructs targeting Gprk2 expression in vivo. This led to an unexpected and novel discovery. Even though blood cells give rise to the most apparent phenotypes in the Tl10b larva, the main source for the immune signal is the fat body. This indicates that besides the humoral response, also in cell based immunity this organ plays a major role. Based on this finding, I could show that the modification of Tl10b blood cell phenotypes caused by loss of ird1 expression are due the role this gene plays in autophagy cell motility. The improved understanding of these basic and evolutionary highly conserved mechanisms will undoubtedly help in fending off infectious disease in both man and honeybees in the future.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 81 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1670
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-92751 (URN)978-91-7601-116-4 (ISBN)
Public defence
2014-09-26, Major Groove, building 6L, molekylärbiologi, Umeå University, Umeå, 09:00 (English)
Available from: 2014-09-05 Created: 2014-09-02 Last updated: 2014-09-08Bibliographically approved

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Schmid, Martin RudolfHultmark, Dan
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