Drosophila melanogaster Rac2 is necessary for a proper cellular immune response
M 8O 10 Rac2 riginal J Williams regulates Article ethemocyte al. Ltd. activation Blackwell Oxford, Genes GTC © ?1365-2443 2005 Blackwell toUK Cells Publishing, Publishing Ltd
Michael J. Williams1,*, Istvan Ando2 and Dan Hultmark1 1
Umeå Centre for Molecular Pathogenesis (UCMP), Umeå University, S-901 87, Umeå, Sweden Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, PO Box 521, Hungary
2
It has been reported that during Drosophila embryonic development, and in cell culture, that the Rac GTPases are redundant. To better elucidate Rac function in Drosophila, we decided to study the role of Rac2 in larval cellular defense reactions against the parasitiod Leptopilina boulardi. Here we show a dramatic effect in the context of cellular immunity where, unlike embryonic development, Rac2 appears to have a non-redundant function. When an invading parasitoid is recognized as foreign, circulating hemocytes (blood cells) should recognize and attach to the egg chorion. After attachment the hemocytes should then spread to form a multilayered capsule surrounding the invader. In Rac2 mutants this process is disrupted. Immune surveillance cells, known as plasmatocytes, adhere to the parasitoid egg but fail to spread, and septate junctions do not assemble, possibly due to mislocalization of the Protein 4.1 homolog Coracle. Finally, larger cells known as lamellocytes attach to the capsule but also fail to spread, and there is a lack of melanization. From these results it appears that Rac2 is necessary for the larval cellular immune response.
Introduction The blood cells (hemocytes) of Drosophila play a key role in immune surveillance and are active against pathogens and parasites.When the morphology of circulating blood cells is compared, three basic types of cells can be identified. The most abundant cells are the plasmatocytes.These are small cells involved in phagocytosis and encapsulation, as well as producing anti-microbial peptides.The largest and least abundant cells are the lamellocytes, non-phagocytic specialized spread cells not normally found in healthy larvae. Plasmatocytes, lamellocytes and crystal cells, the third hemocyte cell type, are involved in the encapsulation of invading pathogens (Meister 2004). Crystal cells secrete components of the phenol oxidase cascade involved in melanization. When an invading organism is recognized as foreign it should be removed rapidly by phagocytosis and/or encapsulation by circulating hemocytes.This reaction can be observed when the parasitoid wasp Leptopilina boulardi lays its eggs in the hemocoel of second instar Drosophila larvae.This invasion elicits a strong cellular response, with the release of plasmatocytes from a hematopoietic organ Communicated by : Kozo Kaibuchi *Correspondence : E-mail:
[email protected] DOI: 10.1111/j.1365-2443.2005.00883.x © Blackwell Publishing Limited
known as the lymph gland (Lanot et al. 2001) and possibly also a sessile population found throughout the larvae. Furthermore, it causes the differentiation of numerous lamellocytes (Carton & Nappi 1997; Meister & Lagueux 2003; Meister 2004). Once the wasp egg is recognized, capsule formation ensues. This requires circulating plasmatocytes to change from a nonadhesive to adhesive state enabling them to bind to the invader. First the plasmatocytes attach and spread around the chorion of the wasp egg.After the cells have spread they form septate junctions, thus separating the egg from the hemocoel. The last phases of capsule formation include lamellocyte adherence, and melanization due to crystal cell degranulation (Russo et al. 1996). From these encapsulation events it is obvious that adhesion and cell shape change is an essential part of the cellular response against parasitoid wasp eggs. One family of proteins central to the processes involved in cell shape are the Rac GTPases (Burridge & Wennerberg 2004; Raftopoulou & Hall 2004). Once activated, Racs are involved in many cellular processes including: cytoskeletal organization, the regulation of cellular adhesion, cellular polarity, and transcriptional activation (Burridge & Wennerberg 2004). The challenge is to understand the spatial and temporal regulation of the signaling events involved in this cellular response. Genes to Cells (2005) 10, 813–823
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The Drosophila genome encodes three Rac GTPases: Rac1, Rac2 and Mig-2-like (Mtl). It has been reported during development and in cell culture that the three Drosophila Racs are redundant (Hakeda-Suzuki et al. 2002; Kunda et al. 2003; Ng et al. 2002; Rogers et al. 2003). Sasamura et al. (1997) and the Berkeley Drosophila Genome Project have reported that Rac2 (called Drac1b) mRNA is expressed in embryonic hematopoietic tissues. Sasamura also reported that Drac3 was expressed in hemocytes, but since their report Drac3 has been reclassified as RhoL a non-conical Rho GTPase. Later, Boutros et al. (2002) found that Rac2 was up-regulated in S2 cells upon LPS addition. This gave us the idea to examine if Rac2 has a function in larval hemocytes during immune surveillance. In order to understand the function of Rac2 GTPase in regulating the cellular immune response we have assayed the effects on hemocytes in parasitized Rac2 mutant larvae, as well as characterized the effect of overexpressing Rac2 in hemocytes.We report here that Rac2 has an important role in the cellular immune response, being necessary for hemocyte spreading and cell-cell contact formation during immune surveillance against the parasitoid L. boulardi.
Results Wasp encapsulation
A darkened cellular capsule surrounding a parasitoid wasp egg was easily visible in the hemocoel of wild-type Drosophila larvae 30– 40 h after parasitization by the avirulent L. boulardi wasp strain G486. We used this as the basis for a wasp encapsulation assay to test how Rac2 loss-of-function mutants affect the cellular immune reaction (Sorrentino et al. 2002). Since genetic variability in the genomic background can affect this response, two independently created Rac2 alleles were used, Rac2∆ and Rac2[KG05681b]. Rac2∆ is a P element imprecise excision resulting in a deletion that removes most of the Rac2 open reading frame, creating a null allele (Ng et al. 2002). Rac2[KG05681b] has a P element contained within the open reading frame of Rac2 that disrupts the gene (isolated by the Berkeley Drosophila Genome Project). In Rac2∆/TM6,Tb control larvae 40– 42 h after parasitization 71.2% of the wasp eggs were correctly encapsulated, in Rac2∆ homozygotes the rate of proper encapsulation was 0% (Fig. 1). Rac2[KG05681b]/ TM6,Tb heterozygotes properly encapsulated 89.4% of the wasp eggs, while in Rac2[KG05681b] homozygotes, similar to Rac2∆ homozygotes, the rate of proper encapsulation was also 0% (Fig. 1). This was also the case with Rac2∆/Rac2[KG05681b] transheterozygous larvae (Fig. 1). From this we conclude that Rac2 function is necessary for proper 814 Genes to Cells (2005) 10, 813– 823
Figure 1 Encapsulation of wasp eggs in control and Rac2 mutant larvae. Encapsulation capacities of Rac2 mutant larvae in response to parasitization by L. boulardi G486. Numerical values for proper encapsulation percentages [(Number of properly melanized wasp eggs/number of parasitized larvae) × (100)] are presented above each bar. Numbers in parentheses indicate the number of waspparasitized larvae examined.
capsule formation in response to eggs from the parasitoid L. boulardi. Encapsulated wasp eggs dissected from control larvae had an inflated appearance, with a space easily visible between the chorion/hemocyte capsule and the wasp embryo (Fig. 2A). This space between the developing embryo and the chorion/hemocyte capsule was not due to shrinkage of the paraformaldehyde fixed wasp embryo. In equally aged unfixed encapsulated wasp embryos, the space was still present and many of the embryos were seen moving. Although not visible in the living larvae, improperly encapsulated wasp eggs were recovered from Rac2 homozygous mutant larvae. In contrast to wasp eggs dissected from control larvae, in Rac2∆ homozygous mutant larvae darkening of the capsule surrounding the wasp egg and the separation between the wasp embryo and the chorion/hemocyte capsule was never observed (Fig. 2B).The lack of melanization seen in Rac2 mutants could be due to improper capsule formation or due to a lack of crystal cells necessary for melanization of the capsule.To test the latter possibility we heated third instar Rac2 heterozygous and homozygous mutant larvae for 10 min at 60 °C. This activates the prophenoloxidase within the crystal cells causing them to turn black (Rizki & Rizki 1980). We counted the two most posterior segments of at least 15 larvae, and from this assay it was evident that the number of crystal cells in Rac2 mutants © Blackwell Publishing Limited
Rac2 regulates hemocyte activation
Figure 2 Rac2 mutant capsules fail to darken. (A) Encapsulated wasp eggs recovered from Rac2∆/TM6b,Tb 40 h postparasitization. (B) Encapsulated wasp eggs recovered from Rac2∆/Rac2∆ 40 h postparasitization. Crystal cells in Rac2∆/ TM6b,Tb and Rac2∆/Rac2∆ wandering third instar larvae.The posterior part of the larvae are shown for (C) Rac2∆/TM6b,Tb and (D) Rac2∆/Rac2∆. A total of 15 larvae/ genotype were visualized and the crystal cells of the last two posterior segments were counted.
(Fig. 2D; Rac2∆/Rac2∆ = 77 ± 10) was comparable to those found in controls (Fig. 2C; Rac2∆/TM6b,Tb = 85 ± 17). Rac2 is necessary for proper plasmatocyte function
It is known that Rac GTPases regulate actin cytoskeleton dynamics (reviewed by Raftopoulou & Hall 2004; Brunton et al. 2004). To see if Rac2 could be regulating actin cytoskeletal formation, hemocytes were bled from either Rac2∆/Rac2∆ mutants or Rac2∆/TM6b,Tb control larvae before and after parasitization by L. boulardi, and stained with TRITC-phalloidin to visualize the actin cytoskeleton. Hemocytes recovered from control larvae 30 h after parasitization were larger and had intensely ruffled plasma membranes, when compared to hemocytes recovered from non-parasitized larvae (Fig. 3A,C). Hemocytes recovered from parasitized Rac2∆ homozygous mutants were smaller, rounder in appearance, and had less pronounced F-actin staining than equally aged parasitized control hemocytes (Fig. 3C,D). Hemocytes from parasitized Rac2∆ homozygous mutants were also smaller and rounder in appearance than hemocytes recovered from non-parasitized Rac2∆ homozygous mutants (Fig. 3B,D). By 40 h postparasitization, control hemocytes had thick actin filaments at the plasma membrane, and long thin filopodia (Fig. 3E). Hemocytes recovered from parasitized Rac2 mutants at the same time didn’t look any different from hemocytes recovered at 30 h after parasitization (Fig. 3F). To gain further insight into the encapsulation defects in Rac2 mutants, we compared capsules recovered from © Blackwell Publishing Limited
parasitized Rac2∆ and Rac2[KG05681b] heterozygous and homozygous larvae. Second instar Drosophila larvae were parasitized, and then allowed to develop for 24–26 h. After this time the wasp egg was removed from the hemocoel and stained with the plasmatocyte specific α-P1b antibody (Kurucz et al. 2003), to visualize the morphology of the plasmatocytes attached to the chorion. By 24 h postparasitization most of the wasp eggs dissected from control larvae were fully encapsulated by plasmatocytes that had spread around the chorion (Fig. 4A). In these spread plasmatocytes forming the capsule, the plasmatocytespecific P1b antigen was localized to a lateral ring of the plasma membrane (Fig. 4A). This was different from plasmatocytes that had not adhered to the wasp egg, where the P1b antigen was not obviously localized to the plasma membrane, and appeared to be mostly cytoplasmic (Fig. 4C). Plasmatocytes that had attached to the wasp eggs dissected from equally aged Rac2∆ or Rac2[KG05681b] homozygous larvae looked completely different from their heterozygous counterparts. In Rac2 homozygous mutants plasmatocytes adhered to the wasp egg, but failed to spread around it, and the P1b antigen remained in the cytoplasm instead of completely mobilizing to the plasma membrane (Fig. 4B). This led to the idea that Rac2 may somehow play a role in the localization of P1b in plasmatocytes during the encapsulation process.To test this we crossed the Rac2 over-expressing line EP(3)3118 to the hemocyte-specific Hemolectin GAL4 driver (Goto et al. 2003). EP(3)3118 has a UAS P element upstream of Rac2 that was already shown to over-express Rac2 (PenaRangel et al. 2002; Tseng & Hariharan 2002). When Genes to Cells (2005) 10, 813–823
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Figure 3 Actin cytoskeleton in Rac2∆/ TM6b,Tb and Rac2∆/Rac2∆ hemocytes after parasitization. Hemocytes were recovered from non-parasitized larvae (A,B), as well as 30 h (C,D) or 40 h (E,F) after parasitization and the actin cytoskeleton was visualized using TRITC-phalloidin, the nucleus is stained with DAPI. (A,C,E) Rac2∆/TM6b,Tb (B,D,F) Rac2∆/Rac2∆.
Figure 4 Plasmatocytes fail to spread in homozygous Rac2 mutants. (A,B) Encapsulated wasp eggs recovered from larvae 24–26 h after parasitization, and stained with the plasmatocyte specific antibody P1b. (A) Rac2∆/TM6b,Tb. (B) Rac2∆/Rac2∆. (C,D) Bled plasmatocytes from third instar larvae, and stained with the plasmatocyte specific antibody P1b, the nucleus is stained with DAPI. (C) EP(3)3118/EP(3)3118 control hemocytes. (D) HML-GAL4; EP(3)3118 hemocytes over-expressing Rac2.
Rac2 was over-expressed in hemocytes the antigen P1b completely localized to the plasma membrane in circulating plasmatocytes (Fig. 4D). This, along with the lack of P1b recruitment to the plasma membrane in a Rac2 816 Genes to Cells (2005) 10, 813– 823
mutant, shows that Rac2 is involved in regulating P1b antigen expression at the plasma membrane after plasmatocytes have recognized and spread on the chorion of the wasp egg. © Blackwell Publishing Limited
Rac2 regulates hemocyte activation
Figure 5 Coracle expression in Rac2∆/ TM6b,Tb and Rac2∆/Rac2∆ 30 h postparasitization. (A) Encapsulated wasp eggs recovered from Rac2∆/TM6b,Tb and stained for Coracle. Inset: Three plasmatocytes are shown with Coracle at the plasma membrane. Newly attached plasmatocytes have Coracle in the cytoplasm (arrowhead). (B) Encapsulated wasp eggs recovered Rac2∆/ Rac2∆. Inset: Four plasmatocytes are shown with Coracle expression mostly perinuclear. (C-F) Bled plasmatocytes from non-parasitized larvae or 30 h after parasitization, and stained α-Coracle, the nucleus is stained with DAPI. (C) Non-parasitized Rac2∆/TM6b,Tb. (D) Parasitized Rac2∆/TM6b,Tb. (E) Nonparasitized Rac2∆/Rac2∆. (F) Parasitized Rac2∆/Rac2∆.
Previously we showed that over-expression of Rac1 GTPase in hemocytes caused overproliferation of plasmatocytes and differentiation of lamellocytes (Zettervall et al. 2004).When Rac2 was over-expressed there was an effect on P1b localization, yet there was no induction of plasmatocyte proliferation or lamellocyte differentiation. Also, the over-expression of Rac1 in hemocytes does not influence the cellular distribution of the P1b antigen (M. Williams, unpublished observation).We feel these results show that Rac1 and Rac2 have differing roles in larval cellular immune activation. Septate junctions fail to form in Rac2 mutants
After the hemocytes attach and spread over the wasp egg, they form septate junctions (Russo et al. 1996). Septate junctions are thought to be analogous to the vertebrate paranodal septate junction (Bellen et al. 1998), and should form a barrier between the wasp egg and the Drosophila hemocoel. One protein known to be critical for septate juction formation is the Drosophila Protein 4.1 homolog Coracle (Fehon et al. 1994). We used Coracle antibody to see if septate junction formation was affected in Rac2 © Blackwell Publishing Limited
mutants. In wasp eggs dissected from Rac2 heterozygous control larvae 25 h postparasitization, no septate junctions were visible using the Coracle antibody as a marker (data not shown). By 30 h postparasitization septate junctions were seen between some plasmatocytes that had attached and spread around the wasp egg (Fig. 5A, see inset).While other more rounded cells, as if they had just attached to the chorion, had Coracle expression throughout their cytoplasm and no septate junctions had yet formed (Fig. 5A, arrowhead). In eggs dissected from Rac2∆ or Rac2[KG05681b] homozygous mutant larvae plasmatocytes were visibly attached to the wasp egg at 30 h postparasitization, but similar to what was seen at earlier time points the cells had not spread around the chorion (Fig. 5B). In most of these cells no Coracle expression was visible, but in the few where Coracle was still expressed, the protein was cytoplasmic instead of at the plasma membrane and no septate junctions were visible (Fig. 5B, see inset). In Rac2 mutants most of the plasmatocytes attached to the wasp egg lack Coracle expression. Because of this we decided to look at its expression in circulating hemocytes. Coracle is expressed in the cytoplasm of plasmatocytes of non-parasitized heterozygous control and Rac2 Genes to Cells (2005) 10, 813–823
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homozygous mutant larvae (Fig. 5C,E).While the pattern of expression in plasmatocytes of control larvae had not changed 30 h after parasitization (Fig. 5D), its expression had decreased in lamellocytes (data not shown). Some plasmatocytes bled from parasitized Rac2 mutants 30 h after parasitization had perinuclear Coracle expression, though in most the expression had completely disappeared (Fig. 5F).As seen with a few cells attached to the wasp egg, some circulating cells still expressed Coracle throughout the cytoplasm (data not shown).We conclude that septate junctions fail to form in Rac2 mutants, possibly due to a lack of cell spreading caused by abnormalities in the actin cytoskeleton, as well as an inability to maintain Coracle expression after parasitization. Lamellocytes are affected by loss of Rac2
After plasmatocytes have surrounded the wasp egg, and formed septate junctions, lamellocytes adhere to the egg, completing capsule formation (Russo et al. 1996). We speculated that disruption of earlier encapsulation events might inhibit lamellocyte function.To study this, capsules were recovered from parasitized control larvae 40 h after parasitization, and stained with the lamellocyte specific antibody L1 (Kurucz et al. 2003). By 40 h after parasitization, spread lamellocytes were observed completely surrounding the egg (Fig. 6A).Wasp eggs were also recov-
ered from Rac2∆ or Rac2[KG05681b] homozygotes 40– 42 h after parasitization, and stained with the L1 antibody. Similar to what we saw with plasmatocytes, lamellocytes had adhered to the egg in a rounded form instead of their usual spread appearance (Fig. 6B; compare Fig. 6B with 6D). At this same time we observed that circulating lamellocytes from control and Rac2 mutant larvae, had spread on the glass surface of the slide (Fig. 6C,D). This means that lamellocytes that are deficient for Rac2 have the ability to take on the normal spread morphology, but show an abnormal rounded appearance when attached to the capsule surrounding the wasp egg.
Discussion In Drosophila, study of Rac GTPases has shown their involvement in regulating many developmental events including actin-mediated cell shape changes (reviewed by Settleman 2001). Until recently most of these studies were done with various Rac over-expression constructs.To look at the lossof-function phenotypes of the different Racs found in Drosophila, Ng et al. (2002) created mutations in all three Racs and showed that Rac2 nulls had weak axon guidance defects. Hakeda-Suzuki et al. (2002) characterized the same mutations in dorsal closure and myoblast fusion, and also found that Rac2 mutants had subtle defects. Furthermore, Paladi & Tepass (2004) showed that Rac1 and Rac2 were
Figure 6 Lamellocyte morphology is disrupted in Rac2 mutants. Hemocytes 40 h postparasitization stained with the L1 lamellocyte specific antibody. (A) Encapsulated wasp egg recovered from Rac2∆/TM6b,Tb 40 h postparasitization. (B) Encapsulated wasp egg recovered from Rac2∆/Rac2∆ 40 h postparasitization. (C,D) Lamellocytes 40 h postparasitization stained with the L1 lamellocyte specific antibody. (C) Rac2∆/TM6b,Tb (D) Rac2∆/ Rac2∆.
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redundant, and Mtl was not necessary, during embryonic hemocyte migration.Yet, when LPS is added to Drosophila S2 cells, of the three Racs only Rac2 is transcriptionally up-regulated (Boutros et al. 2002). Also, in a microarray to look at genes that were enriched in larval hematopoietic tissues, again of the three Racs only Rac2 was transcriptionally enriched (Irving et al. 2005).This leads to the idea that Rac2 must have important functions other than during Drosophila embryonic development. Since Rac GTPases are known to regulate actin cytoskeletal formation, it was not surprising that Rac2 homozygous mutants had defects in actin cytoskeleton rearrangements. In parasitized control larvae hemocytes have extensive membrane ruffling, as well as long thin filopodia all over the cell. This is never observed in hemocytes bled from parasitized Rac2 homozygous mutants. It is believed that the Rho GTPase family member Cdc42 is responsible for inducing filopodial formation (Miki et al. 1998). The lack of filopodia in Rac2 mutants could mean Rac2 signals upstream of Cdc42 in hemocytes. There is another intriguing possibility. Filopodia formed by the actions of Cdc42 are usually relatively short and arise at the cell periphery (Miki et al. 1998; Pellegrin & Mellor 2005).The filopodia found on activated hemocytes are long and found not only at the cell periphery, but on the apical surface as well. This is more reminiscent of Rif GTPase induced flilopodia found on mammalian cells (Pellegrin & Mello 2005). Though no Rif ortholog exists in Drosophila, it is related to Drosophila Rac2. The lack of long apical filopodia in Rac2 mutants leads to the possibility that Rac2 is acting like Rif GTPase in activated hemocytes. Whether Rac2 alone can induce filopodia, or requires the actions of Cdc42 has yet to be elucidated. Another interesting observation was that hemocytes recovered from parasitized Rac2 homozygous mutants were smaller than hemocytes from non-parasitized Rac2 mutants. Sander et al. (1999) showed that Rac1 was involved in inhibiting Rho1 activation in NIH3T3 fibroblasts.The smaller cell size, and lack of membrane ruffling seen in hemocytes recovered from parasitized Rac2 mutants, could be due to an overactivation of Rho1 GTPase. This would lead to a higher amount of stress fiber formation and thus a smaller cell size.When the actin cytoskeleton of plasmatocytes bled from parasitized Rac2 homozygous mutants is compared with that of plasmatocytes from heterozygous Rac2 larvae, it is evident that Rac2 is necessary for actin remodelling after cellular immune activation. The lack of susequent cellular spreading in Rac2 mutants may also be a consequence of adhesion molecules not being properly distributed to the plasma membrane.The antibody P1b used in this study comes from a library of © Blackwell Publishing Limited
monoclonal antibodies raised against hemocyte-specific membrane proteins (Kurucz et al. 2003). P1b does recognize a plasmatocyte-specific plasma membrane epitope (I. Ando, unpublished data). In this study P1b was observed in the cytoplasm of activated circulating hemocytes, and was fully recruited to the plasma membrane of plasmatocytes only when they had adhered and spread on the wasp egg. This translocation requires the activity of Rac2, since in Rac2 mutants P1b is still seen throughout the cytoplasm, and is always found at the plasma membrane of hemocytes from Rac2 over-expressing larvae. This is evidence that Rac2 is regulating the cellular location of at least one plasma membrane molecule, possibly by regulating vesicular trafficking. If Rac2 is regulating vesicular trafficking, this may have an effect on cellular adhesion. Although the precise function of vesicular trafficking in cellular adhesion is unclear, the accumulating evidence shows that facilitated recycling of cell adhesion molecules is important for membrane protrusion (Symons & Rusk 2003). Cellular spreading would require the same recruitment of integrins to the leading edge of the growing protrusions. It is therefore conceivable that Rac2 GTPase could regulate cellular spreading by coordinating both vesicular trafficking and actin dynamics. Though no evidence has been found for the involvement of Rac GTPases in septate junction formation or maintenance, they are known to be active in adherence junctions (Braga 2002; Symons & Rusk 2003). Expression of constitutively active Rac1 in MDCK cells increased actin filaments at cell-cell adhesion sites, while expression of a dominant negative form of Rac1 in these cells decreased adhesion sites (Takaishi et al. 1997). Furthermore Lambert et al. (2002) showed that Rac1 GTPase was necessary for cadherin binding to the actin cytoskeleton during the formation of adherens junctions. This is evidence that Rac GTPase has a function in the formation of at least one type of cellular junction. In parasitized Rac2 mutants the septate junction protein Coracle fails to be recruited to the plasma membrane. This mislocalization may lead to its degradation, and be the cause of the lack of expression we see in Rac2 deficient hemocytes.There is a problem with this model. In parasitized control larvae 30 h postparasitization, Coracle protein is still cytoplasmic in circulating plasmatocytes, only becoming localized to the plasma membrane in cells that have spread on the wasp egg, and adhered to one another. In Rac2 deficient hemocytes Coracle is reduced or absent after parasitization, which suggests that after activation Rac2 is somehow necessary to maintain Coracle expression, as well as for its proper localization in activated hemocytes. It is possible that when plasmatocytes are activated by parasitization, Rac2 Genes to Cells (2005) 10, 813–823
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is involved in recruiting Coracle to complexes on cytoplasmic vesicles for transfer to septate junctions, thus protecting Coracle from degradation. By 40 h after parasitization of control larvae, multiple layers of spread plasmatocytes and lamellocytes are observed attached to the wasp egg. In Rac2 mutants the lamellocytes still adhere to the capsule, but with a small and rounded morphology.This could be due to the malformed underlying plasmatocyte layer. For lamellocytes to maintain their spread appearance they must be critically dependent on mechanical force production by the cytoskeleton. Regulation of cellular processes by mechanical force, mechanosensitivity, has been established in several contexts. Studies in tissue culture cells have revealed tension-sensitive signaling events (Riveline et al. 2001; Suter & Forscher 2001). Furthermore, focal adhesion complexes (cell-matrix interaction) and adherence junctions (cell-cell interaction) have been argued to function as loci of mechanosensing (Ko & McCulloch 2001; Bershadsky et al. 2003). Application of external mechanical force can also change gene expression and cause cytoskeletal changes. The expectation would be that the cellular dynamics of a lamellocyte in suspension versus one that has adhered to plasmatocytes during encapsulation would differ. Thus, lamellocytes might be able to maintain their spread appearance in suspension, but when they interact with plasmatocytes may require other signaling events or cell-cell interactions to maintain their morphology. In Rac2 mutants the plasmatocytes do not form a contiguous sheet for the lamellocytes to adhere to and cell-cell junctions are absent; this may disrupt mechanical force production by the cytoskeleton causing them to round up. We cannot rule out the
possibility that functional Rac2 is required in the lamellocytes during the encapsulation process, in a cell autonomous manner, for them to maintain a spread morphology. Melanization is another event disrupted in capsules recovered from Rac2 mutants. After the capsule forms it normally darkens due to melanization (Russo et al. 1996; Carton & Nappi 1997; Meister 2004). There should also be killing of the wasp embryo, believed to be due to asphyxiation and/or by the production of cytotoxic free radicals, quinones or semiquinones within the wasp egg (Nappi et al. 1995, 2000). In Rac2 mutants there are wild-type numbers of crystal cells, but no darkening of the encapsulated wasp egg. This could mean that crystal cell function is disrupted, or that proper capsule formation may be necessary to trap the proteins needed for the production of melanin at the wasp egg. It could also mean that a signal is required from the properly formed capsule itself to activate melanization. These possibilities will have to be elucidated in future. Finally, the lack of space seen between the capsule and the wasp embryo in Rac2 mutants, normally seen in controls, could reflect the fact that the hemocytes have not formed a contiguous sheath, sealing the parasite from the hemolymph. This could further lead to a lack of cytotoxic free radicals, quinones or semiquinones production by the cells in the capsule.All of this put together leads to the survival of the wasp in Rac2 mutants. Rac GTPases are highly conserved throughout evolution. We attempted to deduce the phylogenetic relationship between the various Rac proteins by performing maximum parsimony analysis (Swofford 1991) (Fig. 7). From this tree two observations can be inferred. First, there were two separate duplication events leading to Drosophila and
Figure 7 Phylogenetic tree of the Rac genes. A maximum parsimony tree was constructed with the amino acid sequences, by using the paup program (Swofford 1991). For branches supported by bootstrap analysis, the percentage of 1000 replications that support the branch is indicated.
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mammalian Rac1 and Rac2 (Fig. 7). Second, even though Racs are evolving at an extremely low rate, it is evident that the Drosophila and mammalian Rac2s are evolving at a faster rate than their Rac1 counterparts (Fig. 7).All three Drosophila Rac1s are 100% identical. When the three Drosophila Rac2s are compared, Drosophila psueodobscura is evolving away from the other two Drosophilids represented. The same can be said of the mammalian Racs. Human, mouse and rat Rac1 proteins are 100% identical, whereas the human Rac2 differs from the mouse and rat Rac2 proteins. This might be explained by the differing roles the two Racs have during development and immunity (Sasamura et al. 1997; Gu et al. 2003). Rac2’s recruitment for a role in the immune response may actually necessitate its faster evolutionary rate. Mammalian Rac2 is necessary for the proper motility and adhesion of neutrophils (Roberts et al. 1999; Li et al. 2002). In Rac2 deficient mice, neutrophils fail to migrate properly, and cell spreading after integrin ligation is severely reduced (Roberts et al. 1999).This is similar to what is seen in Drosophila Rac2 mutants after parasitization by L. boulardi. In Rac2 homozygous mutants plasmatocytes adhere to the wasp egg but fail to undergo cellular spreading.At present we do not know if this is due to a problem with integrin ligation, but we do show that actin remodelling after immune activation is disrupted. In any case the non-redundant function of Rac2 in the Drosophila immune response is at least superficially similar to the non-redundant function of mammalian Rac2 in the immune system. In summary we believe that Rac2 function is required early during the formation of the capsule. Plasmatocytes still adhere to the egg, showing that they are able to recognize something on the egg for attachment. After adhering they fail to undergo transformation from round cells to an extremely spread morphology. This block in cellular spreading disrupts all later events in capsule formation. These data show that, unlike embryonic development, Rac2 function is not redundant and is necessary during the cellular immune response. Rac2 signaling is needed not only for proper plasmatocyte function, but lamellocyte function as well. It also shows that proper capsule formation is required for darkening of the capsule. Lastly, proper encapsulation is necessary for an inflated morphology of the capsule; the cause or function of this inflation is not yet known.
Experimental procedures Insects Drosophila strains Rac2∆ ry506/Rac2∆ ry506, rac1J11Rac2∆ FRT2A/ TM6b,TB, y1 w67c23; P{y[+mDint2] w[BR.E.BR] = SUPor-
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p}KG05681a; P{SUPor}Rac2[KG05681b]ry506 and w1118; P{w[+mC] = Hml-GAL4.G}6-4 P{w[+mC] = UAS-GFP::lacZ.nls}15.1 were obtained from the Bloomington Stock Center, and the references are given in Flybase (http://fbserver.gen.cam.ac.uk:7081). EP(3)3118 was provided by the stock center at Szeged, Hungary. Flies were kept on a standard mash-potato diet at between 21 and 25 °C. Stocks crossed to Hemolectin-Gal4 driver flies, and the uncrossed control flies, were raised at 29 °C. The G486 strain of L. boulardi was bred on a CantonS stock of D. melanogaster at room temperature using a standard medium. Adult wasps were maintained at room temperature on apple juice plates.
Immunofluorescence For lamellocyte monoclonal antibody (L1a), Plasmatocyte specific monoclonal antibody (P1b) (Kurucz et al. 2003) and the polyclonal Coracle antibody (Fehon et al. 1994), hemocytes and wasp eggs were bled from larvae, into 20 µL of PBS, and allowed to attach to a glass slide (SM-011, Hendley-Essex, Essex, UK) for 1 h (hemocyte only experiments) or 5 min (wasp egg experiments) at room temperature. Stainings and analysis were done according to Zettervall et al. (2004). The lamellocyte monoclonal antibody (L1a) and the plasmatocyte specific monoclonal antibody (P1b) were used undiluted (Kurucz et al. 2003). The polyclonal Coracle antibody (a gift from Rick Fehon) was diluted 1 : 1000 in 3% BSA/PBSA. For F-actin visualization hemocytes were bled from larvae as stated above. The cells were then fixed for 5 min with 3.7% paraformaldehyde/PBS, before being washed once for 5 min with PBS, followed by a 5 min wash with PBST (PBS containing 0.1% of Triton X-100), and a final wash of 5 min with PBS. The cells were then stained for 40 min at room temperature with TRITC-phalloidin (Sigma) diluted to a final concentration of 10 µg/mL in PBS. After this the cells were washed twice for 5 min with PBS, once for 5 min with PBS containing DAPI 1 : 5000, and then a final wash for 5 min with PBS.The cells were then mounted using 50% glycerol in PBS. All cells were visualized using epifluorescence (Axioplan 2 microscope, Zeiss) and digital pictures were taken with a Hamamatsu (C4742-95 video unit, controlled by the Openlab program (Improvision, Coventry, UK). Photoshop (Version 7.0, Adobe Systems, San Jose, CA, USA) was used for digital editing.
Wasp egg encapsulation assay The encapsulation assay was done according to Sorrentino et al. (2002). Briefly, two days before parasitization the appropriate fly strains were crossed and kept at 21–25 °C. Four or five females of L. boulardi G486 were allowed to infest at room temperature for 2 h, after which the Drosophila larvae were transferred to apple juice plates and left at room temperature for 40– 42 h. After this time the larvae were collected, washed in PBS, and then viewed under a stereomicroscope for the presence of a dark capsule. Larvae in which no dark capsule was observed were dissected in 20 µL of PBS to determine if they had been parasitized. Larvae containing eggs from the parasitoid that hadn’t darkened by this time were scored as nonencapsulated. Non-parasitized larvae were excluded from the count. Genes to Cells (2005) 10, 813–823
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Crystal cell counts To visualize crystal cells larvae were heated for 10 min at 60 °C in a water bath (Rizki & Rizki 1980). Only the sessile population of crystal cells in the two most posterior segments were counted (Duvic et al. 2002). At least 15 wandering third instar larvae were counted for each genotype.
Acknowledgements We thank Rick Fehon for his kind gift of Coracle antibody, the Bloomington Stock Center and the stock center at Szeged, Hungary for providing fly stocks, and Shannon Albright for her wasp expertise. This research was supported by grants from the Swedish Research Council, the Swedish Cancer Society, and the Wallenberg Consortium North.
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