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Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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2004 (English)In: Current Biology, ISSN 0960-9822, Vol. 14, 625-629 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 14, 625-629 p.
Identifiers
URN: urn:nbn:se:su:diva-14030DOI: 10.1016/j.cub.2004.03.030OAI: oai:DiVA.org:su-14030DiVA: diva2:180550
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2010-01-14Bibliographically approved
In thesis
1. Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster
Open this publication in new window or tab >>Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies.

By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture.

The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug.

We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2007. 1-30, 36-49 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-7046 (URN)978-91-7155-497-0 (ISBN)
Public defence
2007-09-28, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00 (English)
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Supervisors
Note
At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper 5: ManuscriptAvailable from: 2007-09-06 Created: 2007-08-31 Last updated: 2012-02-15Bibliographically approved
2. Proteomics of the Drosophila hemolymph clot and the function of transglutaminase
Open this publication in new window or tab >>Proteomics of the Drosophila hemolymph clot and the function of transglutaminase
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects rely on a fast and effective coagulation and wound response to avoid loss of body fluids and immobilize pathogens. Arthropod coagulation is in some respect equivalent to vertebrate coagulation but most factors and the regulation of coagulation systems seem not to be phylogenetically conserved. To get a more complete picture of insect clotting we studied the molecular and functional nature of Drosophila hemolymph coagulation.

We developed new proteomic methods to collect Drosophila clotting factors. Several candidate factors were identified, including both predicted and novel clot proteins. Five putative TG (transglutaminase) substrates were found and we could also demonstrate that the clot is involved in immobilization of bacteria. Further investigating the role of TG we found TG to be important for Drosophila coagulation and that Fondue is a major substrate of the enzyme. Using fon RNAi knockdown we showed that Fondue affects the physical properties of the clot. A fon-GFP fusion construct was generated to follow its expression. The cuticle and the clot were labelled suggesting that Fondue is incorporated into both cuticle and clot. Clot properties and composition were affected by inhibiting TG chemically (MDC) and genetically (RNAi). Moreover, interaction between Fondue and Eig71Ee was demonstrated. Previous results indicated that coagulation could have an immune function. In hemolymph preparations, containing selected microorganisms, small deposits were seen on the microbial surfaces. The contents of these were investigated, revealing the presence of procoagulants. The targeting of microbes is instant and depends on TG and its substrates. Entomopathogenic nematode infections were performed to validate the functional importance of TG. TG RNAi knockdown larvae showed increased mortality, supporting an immune function for TG. Altogether, our data provide a more comprehensive picture of Drosophila immunity, and may further improve the understanding of innate immunity in general.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2009. 31 p.
Keyword
Drosophila, clotting, transglutaminase, innate immunity
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-26382 (URN)978-91-7155-847-3 (ISBN)
Public defence
2009-05-08, De Geer-salen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2009-04-16 Created: 2009-03-23 Last updated: 2010-01-14Bibliographically approved

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