title: Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method.
abstract: Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.
authors.affiliation: Department of Molecular Biology and Functional Genomics, University of Stockholm, 10691 Stockholm, Sweden.
authors.forename: Christoph
authors.initials: C
authors.lastname: Scherfer
authors.forename: Christine
authors.initials: C
authors.lastname: Karlsson
authors.forename: Olga
authors.initials: O
authors.lastname: Loseva
authors.forename: Gawa
authors.initials: G
authors.lastname: Bidla
authors.forename: Akira
authors.initials: A
authors.lastname: Goto
authors.forename: Johanna
authors.initials: J
authors.lastname: Havemann
authors.forename: Mitchell S
authors.initials: MS
authors.lastname: Dushay
authors.forename: Ulrich
authors.initials: U
authors.lastname: Theopold
mesh_terms: D000818:Animals; D018407:Bacterial Physiological Phenomena; D001777:Blood Coagulation / Q000502:physiology*; D015153:Blotting, Western; D029721:Drosophila Proteins / Q000097:blood; D004331:Drosophila melanogaster / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000097:blood; D006458:Hemolymph / Q000378:metabolism*; D037102:Lectins / Q000097:blood; D013058:Mass Spectrometry; D008863:Microspheres; D014442:Monophenol Monooxygenase
chemical_list: D029721:Drosophila Proteins; D016326:Extracellular Matrix Proteins; C476120:Hml protein, Drosophila; D037102:Lectins; C089678:Tig protein, Drosophila; D014442:Monophenol Monooxygenase