Volume 14, Issue 7, Pages 625-629 (April 2004) Isolation and Characterization of Hemolymph Clotting Factors in Drosophila melanogaster by a Pullout Method  Christoph Scherfer, Christine Karlsson, Olga Loseva, Gawa Bidla, Akira Goto, Johanna Havemann, Mitchell S Dushay, Ulrich Theopold  Current Biology  Volume 14, Issue 7, Pages 625-629 (April 2004) DOI: 10.1016/j.cub.2004.03.030

Figure 1 The Drosophila Hemolymph Clot Entraps Bacteria (A–C) Drosophila hemolymph clotting. (A) Hemolymph from eight wandering larvae was bled onto a well (4 mm diameter), and the increase in viscosity and formation of strands was demonstrated by drawing the clot out with a needle. (B and C) Phase-contrast views of the center of the resulting fiber (B) and the base (C). (D–F) Labeling of the Drosophila clot and entrapment of bacteria. Hemolymph was bled onto a drop containing GFP-labeled bacteria. (D) Nomarski exposure of the section. (E) The same section labeled with PNA visualized under UV light (the arrows indicate hemocytes, the asterisks fat body fragments). (F) GFP-labeled bacteria (see Supplemental Data) are visualized by green fluorescence. The figure shows results from using gram-negative bacteria, but similar results were obtained with gram-positive bacteria. (G) PNA-labeled clot preparation performed in the absence of bacteria (hemocytes indicated by arrows). (H) A preparation of the clot after lysis of hemocytes (with 0.5% Igepal, Sigma; note that the fibrous strands remain intact). The scale bars correspond to 20 μm. Current Biology 2004 14, 625-629DOI: (10.1016/j.cub.2004.03.030)

Figure 2 In Vitro Clotting Leads to Entrapment of Paramagnetic Beads Top: 0, 10, and 20 Drosophila larvae were bled onto paramagnetic beads and then bead aggregation was performed with microscopy (beads are 2.8 μm diameter). A sample taken after addition of five animals was labeled with PNA to visualize clot fibers (PNA, fiber formation is indicated by an arrow). Bead aggregation was also assessed by using hemolymph from homozygous domino mutant larvae (dom/dom), which showed no aggregation and heterozygous control larvae (dom/+), which efficiently aggregated the beads (note that 50% more hemolymph was used for the sample from homozygous larvae). Bottom: clot formation in Bc mutant larvae. Hemolymph from Bc homozygous mutant larvae was prepared as in Figure 2 and labeled with FITC-conjugated PNA. Note the four black cells (found in the Bc mutant) at the upper left. Current Biology 2004 14, 625-629DOI: (10.1016/j.cub.2004.03.030)

Figure 3 Proteins Retrieved by the Pullout Method (A) Proteins binding to the beads were analyzed by using PAGE on 10% gels (pullout) and were Coomassie stained. In addition a plasma sample (before clotting) and serum (after clotting) were analyzed. One protein (a = CG15825) appears both less abundant after clotting and enriched in the pullout fraction. (B) Higher molecular mass proteins (bracket in [A]) were further analyzed on 5% gels (left lane). The supernatant from the first pullout was added to new beads in three subsequent reactions, which were analyzed for their capacity to aggregate beads (aggr), with “++” indicating a strong reaction and “+” indicating a very weak aggregation in the second pullout and on PAGE. Protein bands whose appearance correlated with aggregation are indicated (b, Hemolectin, c and d, two isoforms of Tiggrin). A protein (Apolipophorin I) found in all reactions is marked with an asterisk. (C) Hemolectin reacts with PNA. Proteins from a pullout assay were analyzed with 5% PAGE and stained with Coomassie (Co) as well as blotted and labeled with PNA. (D) Absence of bead aggregation in hml RNAi knockdown mutants. Bead aggregation was performed as in Figure 2 by using hemolymph from larvae bearing both a UAS hml RNAi construct and an Actin-GAL4 driver (hml RNAi) and control nonexpressing larvae bearing only the UAS hml RNAi construct without the driver (control). Current Biology 2004 14, 625-629DOI: (10.1016/j.cub.2004.03.030)

Figure 4 Proteins Recovered from the PNA-Enhanced Pullout Proteins recovered from the pullout were separated by PAGE on a 10% gel. They include: lsp1γ, larval serum protein 1, γ-subunit; fbp1, fat body protein 1, appearing in two fragments; and PO: phenoloxidase (CG8193, see text for further details, note that subunits of the tetrameric PNA are also recovered). The proteins whose presence correlated with the clotting ability of hemolymph (assayed as in Figure 3B) are underlined. The pattern of proteins in the high molecular mass range was the same as for beads without PNA. To test a possible involvement of transglutaminase in crosslinking clot proteins, the transglutaminase inhibitor monodansylcadaverine (MDC) was also included in the reaction. The patterns are identical except for some differences observed in the range of the most abundant hemolymph proteins (70–80 kDa). Current Biology 2004 14, 625-629DOI: (10.1016/j.cub.2004.03.030)