Article Figures & Tables
Figures
- Fig. 1.
Crystal cell activation involves cell rupture. (A-D) Rupture of crystal cells. Crystal cells immediately (A) and at different time points (4, 6 and 8 minutes respectively) after bleeding (B-D). The cell is shown in phase contrast (A,C) and fluorescence (B,D) showing membrane structures labeled with CD8-GFP. Note that the cell membrane ruptures starting at the top of the cell (indicated by the arrow in B) and the crystals dissolve. A fat body fragment adhering to the crystal cell is indicated (*). (E) Activation of PPO in crystal cells can also be achieved by incubating larvae at 65°C for 10 minutes (e.g. Galko and Krasnow, 2004), leading to the appearance of melanotic spots. Phase-contrast microscopy of the melanotic spots after bleeding reveals activation of PPO in the crystals (insets show a single melanized cell (left) and a group of melanized cells (right). Note that crystal cells appear not to rupture and the crystal not dissolved, although PPO is activated. Also, this activation of PPO in the crystal is not enhanced in a Spn27A mutant background (data not shown). Bars, 10 μm.
- Table 1.
Effect of different mutations on PO activation in the clot
Mutant Melanization Reference da-GAL4>UAS-spn27A − lz-GAL4>UAS-spn27A − Bc − Scherfer et al., 2004 lz − Bidla et al., 2005 lz-GAL4>UAS-CG3066-IR + Hmlf03374 +++ GNBP-1 +++ PGRP-SA +++ PGRP-LC +++ PGRP-LE +++ relish +++ spz2 +++ dTAK1 +++ Act5C-GAL4>UAS-bsk-IR − lz-GAL4>UAS-bsk-IR ++ Act5C-GAL4>UAS-hep-IR − he-GAL4>UAS-RhoA.DA − egr1/egr1 + egr3/egr3 ++ egr1/def +/− he-GAL4>UAS-p35 + -
Hemolymph from mutant animals was prepared and analyzed for melanization of the clot. Loss of melanization is indicated by − and different grades of melanization by: +/−, very weak; +, weak; ++, intermediate and +++, normal. Partial melanization with knockout were lz-Gal4 was used may be due to the early transient expression of lz. Melanization of clots from egr1 homozygous mutants was stronger than in transheterozygotes with a deletion uncovering the same region compatible with the idea that egr1 shows some residual activity (see also Fig. 4 for further analysis of egr mutants).
-
- Fig. 2.
Local activation of phenoloxidase in the Drosophila clot. Drosophila hemolymph clots were prepared as described (Bidla et al., 2005) and examined with phase-contrast (A,C,E) and bright field microscopy (B,D,F). Melanization is mostly restricted to cell fragments (asterisks) and folds in the clot matrix (arrowheads indicate clot folds, the arrows indicate plasmatocytes). Bars, 10 μm. (E,F) Plasmatocytes inside the clot (filled arrows) show melanization beginning on their surface. Plasmatocytes outside the clot (open arrow) are not melanized. (G,H) Overexpression of Spn27A (da-GAL4 driven) leads to complete loss of melanization, although clot fibers are formed (visible in phase-contrast, also visible in bright field images G,H). The insets in B and H show PO activity analyzed with a dot-blot assay (see Materials and Methods).
- Fig. 3.
Hemocytes in the clot show hallmarks of apoptosis. Bright field (A) and fluorescence (B) microscopy of a TUNEL assay performed on a clot preparation showing TUNEL-positive (filled arrows) and TUNEL-negative plasmatocytes (open arrows) in the clot. The border of the clot is indicated by arrowheads. (C-E) Phase contrast (C), annexin V (D) and propidium iodide (E) labeling of plasmatocytes 7 minutes after bleeding showing live (*) as well as apoptotic (open arrow, only annexin V positive) and necrotic cells (solid arrow, reacting with both annexin V and propidium iodide).
- Fig. 4.
Melanization defects in egr mutants. (A,B) an egr1/def mutant crystal cell is shown 10 minutes (A) and 60 minutes (B) after bleeding. Crystals are still visible in A (arrows) and not in B without leading to visible melanization. Bar, 10 μm. (C,D) Melanization in egr mutants was measured photometrically. (C) Melanization is reduced in egr1/def mutants compared to wild-type (wt) activity (n=3, *P=0.014). When Egr is overexpressed using he-Gal4 in an egr1 homozygous background melanization is restored to levels above wild-type activity (n=3, **P=0.007 for comparison with the wild type). (D) Melanization in UAS-egr control larvae and in larvae overexpressing Eiger in the fat body (lsp2-Gal4>UAS-egr) or in hemocytes (he-Gal4>UAS-egr). Only expression in hemocytes leads to significant induction (n=3, **P=0.006). Note that the crosses were kept at 25°C. Data are mean ± s.d.
- Fig. 5.
Induction of apoptosis activates PO in vivo in a Spn27A-dependent way. (A) Expression of the apoptotic inducer Grim together with GFP in hemocytes (he-GAL4 UAS-GFP.nls>UAS-grim) leads to formation of melanotic aggregates. Note that he is not expressed in all hemocytes, leaving some cells unaffected. (B) In a spn27a mutant background both the number and severity of the melanotic spots after expressing Grim in hemocytes is increased. (C) Spn27A co-expression with Grim in hemocytes (using the same driver but without UAS-GFPnls to compensate for GAL4 dosage in A and B) abolishes melanotic spot formation almost completely. (D,E) A preparation of an aggregate dissected from a larva such as those shown in A shows the presence of hemocytes which were labeled with GFP. Bar, 10 μm. Note that adults emerging from the cross in Fig. 5A show defects in wing morphology and co-expression of Grim and viral p35 protein abolishes the phenotypes attributable to Grim (not shown).
- Fig. 6.
Drosophila signal transduction pathways involved in immunity and their contribution to crystal cell rupture. Mutants in the factors that are shown in red were tested, mutants that show defects crystal cell rupture are also underlined (see Brennan and Anderson, 2004; Delaney et al., 2006). In addition to regulating the early phase of transcriptional immune and stress responses (Boutros et al., 2002; Park et al., 2004), Bsk acts in a transcription-independent way downstream of Rho GTPases and upstream of cytoskeletal regulators. Dl/Dif, dorsal/dorsal-related immunity factor; GNBP1, gram-negative binding protein 1; IMD, immune deficiency; PGRP-LC, peptidoglycan recognition protein LC; PGRP-LE, peptidoglycan recognition protein LE; PGRP-SA, peptidoglycan recognition protein SA; TAK, HIV Tat-associated kinase; Tl, toll.
Article navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
2020 at The Company of Biologists
Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.
Mole – The Corona Files
"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."
Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.
Cell scientist to watch – Christine Faulkner
In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.
Read & Publish participation extends worldwide
“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”
Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.
JCS and COVID-19
For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.
If you have any questions or concerns, please do not hestiate to contact the Editorial Office.