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Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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. , p. 1-30, 36-49
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-7046ISBN: 978-91-7155-497-0 (print)OAI: oai:DiVA.org:su-7046DiVA, id: diva2:197541
Public defence
2007-09-28, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00 (English)
Opponent
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
List of papers
1. Hemolymph coagulation and phenoloxidase in Drosophila larvae
Open this publication in new window or tab >>Hemolymph coagulation and phenoloxidase in Drosophila larvae
2005 In: Developmental & Comparative Immunology, ISSN 0145-305X, Vol. 29, no 8, p. 669-79Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24441 (URN)
Note
Part of urn:nbn:se:su:diva-7046Available from: 2007-09-06 Created: 2007-08-31Bibliographically approved
2. Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method
Open this publication in new window or tab >>Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method
Show others...
2004 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 14, p. 625-629Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-14030 (URN)10.1016/j.cub.2004.03.030 (DOI)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2017-12-13Bibliographically approved
3. A role for Hemolectin in coagulation and immunity in Drosophila melanogaster
Open this publication in new window or tab >>A role for Hemolectin in coagulation and immunity in Drosophila melanogaster
Show others...
2007 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 31, no 12, p. 1255-1263Article in journal (Refereed) Published
Abstract [en]

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(−) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

Place, publisher, year, edition, pages
Elsevier Ltd, 2007
Keywords
Insect immunity; Innate immunity; Hemolymph coagulation; Discoidin; Lectin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-24443 (URN)10.1016/j.dci.2007.03.012 (DOI)000251491900008 ()
Available from: 2007-09-06 Created: 2007-08-31 Last updated: 2017-12-13Bibliographically approved
4. Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger
Open this publication in new window or tab >>Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger
2007 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 120, no 7, p. 1209-15Article in journal (Refereed) Published
Abstract [en]

The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.

Keywords
innate immunity; phenoloxidase; apoptosis; hemocytes; JNK; TNF
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24444 (URN)10.1242/​jcs.03420 (DOI)000245103900010 ()
Available from: 2007-09-06 Created: 2007-08-31 Last updated: 2017-12-13Bibliographically approved
5. Activation of Insect Phenoloxidase after Injury: Endogenous versus Foreign Elicitors
Open this publication in new window or tab >>Activation of Insect Phenoloxidase after Injury: Endogenous versus Foreign Elicitors
2009 (English)In: Journal of Innate Immunity, ISSN 1662-811X, Vol. 1, no 4, p. 301-308Article in journal (Refereed) Published
Abstract [en]

The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. We tested how PO is activated following injury in 2 insects, i.e. the fruit fly Drosophila melanogaster and the wax moth Galleria mellonella. Rapid PO activation in Drosophila was limited to discrete areas of the hemolymph clot which forms after injury. Surprisingly, unlike systemic PO activation during bacterial sepsis, clot melanization was not sensitive to microbial elicitors in our assay. Instead, Drosophila clot melanization was activated by endogenous signals such as apoptotic cells and was superinduced by phosphatidylserine, a negatively charged phospholipid normally found on the inner surface of the plasma membrane and exposed during apoptosis. In contrast, melanization in G. mellonella hemolymph was stronger and more uniform and was sensitive to peptidoglycan. This shows that both exogenous and endogenous signals can trigger the same immune mechanism in species and context-dependent ways. Our findings have implications for the evolutionary dynamics of immune mechanisms and are in agreement with recent comparisons of insect immune transcriptomes.

Keywords
Coagulation, Comparative immunology, Drosophila melanogaster, Hemostasis, Innate immunity, Insects, Pattern recognition, Sepsis, Wound healing
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-59964 (URN)10.1159/000168009 (DOI)000268091500004 ()
Note
authorCount :4Available from: 2011-08-04 Created: 2011-08-02 Last updated: 2012-02-15Bibliographically approved

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