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First published online 16 January 2007
doi: 10.1242/jcs.03341
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Research Article |
Umeå Centre for Molecular Pathogenesis (UCMP), Umeå University, S-901 87, Umeå, Sweden
* Author for correspondence (e-mail: michael.williams{at}ucmp.umu.se)
Accepted 14 November 2006
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Summary |
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Key words: Rho GTPase, Hemocytes, Innate immunity, Parasitization
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Introduction |
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). Parasitization, by parasitoid wasps for example, induces the release of more hemocytes into circulation from a hematopoietic organ known as the lymph gland (Lanot et al., 2001), as well as from a sessile hemocyte population (Zettervall et al., 2004). Furthermore, after parasitization a third cell type, known as lamellocytes, appears (Carton and Nappi, 1997; Meister, 2004; Meister and Lagueux, 2003). Lamellocytes, rarely seen in healthy larvae, are larger than other hemocytes and are involved in the encapsulation of invading pathogens (Lanot et al., 2001; Sorrentino et al., 2002).
Once a wasp egg is recognized as foreign, the circulating plasmatocytes become more adherent, enabling them to attach to the invader. Septate junctions form between the plasmatocytes once they have spread around the wasp egg. Junction formation effectively separates the wasp egg from the larval hemocoel. The final phases of encapsulation include lamellocyte adherence, and melanization of the capsule as a result of crystal cell degranulation (Russo et al., 1996; Williams et al., 2005). From these events it is obvious that adhesion and cell shape change are an essential part of the cellular immune response against parasitoid wasp eggs.
Rho family GTPases are known to regulate the cytoskeletal rearrangements and adhesions necessary for cell shape change and cellular adhesion (reviewed by Burridge and Wennerberg, 2004; Raftopoulou and Hall, 2004). The Drosophila genome encodes one Rho (Rho1), two Rac (Rac1 and Rac2), one Cdc42 (Cdc42), and three other Rho GTPases: Mig-2-like (similar to mammalian RhoG), Rho-like and RhoBTB. In Drosophila embryos it was shown that the Rho family small GTPases are required for proper plasmatocyte migration during embryogenesis and after wound healing (Paladi and Tepass, 2004; Stramer et al., 2005). The co-ordinated regulation of the various Rho family members is important for directed cell migration, as well as maintenance of cellular adhesions (reviewed by Evers et al., 2000). Rac is required for the formation of broad actin-rich membrane ruffles, known as lamellipodia (reviewed by Ridley, 2001a; Raftopoulou and Hall, 2004; Ridley, 2001b). Cdc42 is necessary for the regulation of cellular polarity and filopodia formation (Allen et al., 1998; Nobes and Hall, 1999). Rho GTPase regulates the formation of contractile actin-myosin filaments that form stress fibers. These stress fibers are necessary to maintain focal adhesions (reviewed by Raftopoulou and Hall, 2004). Rac, as well as Cdc42, has been shown to control focal adhesion turnover (Chrzanowska-Wodnicka and Burridge, 1996; Nobes and Hall, 1995; Nobes and Hall, 1999). From this it is evident that spatiotemporal regulation of the various Rho GTPase family members is important. However, how the various GTPases are spatiotemporally regulated is still largely unknown. There have been reports showing both Rac-induced Rho activation, and Rac inhibition of Rho (Nobes and Hall, 1995; Ridley and Hall, 1992; Sander et al., 1999). There have also been various reports of Rho activating Rac (Salhia et al., 2005; Sinnett-Smith et al., 2001; Tsuji et al., 2002).
In this study we used genetics to elucidate the inter-regulation of Rac1 and Rho1 in Drosophila larval immune surveillance cells. We report here that Rac1, through the Drosophila Jun kinase Basket activates Rho1 and it's downstream effector Rho-kinase. Furthermore, we show that Rho1 can regulate Rac1 activity and that this regulation requires the formin protein Diaphanous. Lastly, though both Rac1 and Rac2 are required for lamellipodia formation after parasitization, only Rac2 is required for filopodia formation.
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; Ng et al., 2002; Williams et al., 2006). As we reported previously (Williams et al., 2006), when hemocytes expressing Rac1F37A were stained to visualize their actin cytoskeleton, unlike controls, thick stress fibers were visible running from the center to the periphery of the cell (Fig. 1A,B). Counting of control (Hemolectin
-Gal4, 2XUAS-eGFP, referred to as Hml-Gal4) and Rac1F37A-expressing hemocytes showed that only 3% of control hemocytes had thick actin stress fibers, compared to 73% of hemocytes expressing Rac1F37A (Fig. 1E).
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; Hickson et al., 2006; Ishizaki et al., 1997; Mizuno et al., 1999; Nakagawa et al., 1996; Watanabe et al., 1999). Depending on the ratio of active Rho-kinase to active Diaphanous, various kinds of stress fibers can be formed (Watanabe et al., 1999). Thick stress fibers are reminiscent of high Rho-kinase activity, whereas thin fibers are indicative of high levels of active Diaphanous (Riento and Ridley, 2003; Watanabe et al., 1999). Since the fibers we observed were relatively thick, possibly indicating high Rho-kinase activity, we co-expressed Rac1F37A with a dominant negative form of Rho-kinase (Rokcatkg). Co-expression led to 83% of hemocytes being round in appearance, the thick actin fibers were replaced by thin fibers, and there was a thick ring of actin at the cell periphery (Fig. 1C,E). This phenotype was never observed in control hemocytes. The thin stress fibers and peripheral actin ring seen in the Rac1F37A and Rokcatkg co-expressing cells are reminiscent of Diaphanous activity (Watanabe et al., 1999).
Since Rac1F37A activates Basket (Bsk) in hemocytes (Williams et al., 2006), it is possible that Rac1 signals through Basket to induce Rho-kinase leading to stress fiber formation (see Fig. 7). To test if Basket was necessary to induce stress fibers we co-expressed Rac1F37A and UAS-bskIR, which expresses a basket-specific double-stranded RNA (Ishimaru et al., 2004). Only a few hemocytes co-expressing Rac1F37A and BskIR had stress fibers and a thick peripheral actin ring (Fig. 1D,E). In fact most cells were very similar to wild-type hemocytes in appearance. This was different from the effect of co-expression of Rac1F37A and dominant negative Rho-kinase in hemocytes, possibly indicating that Basket is necessary for both Rho-kinase and Diaphanous activation. We have already published evidence that overexpression of wild-type Basket in hemocytes does not induce stress fiber formation. Also, the expression of BskIR alone has no obvious effect on the hemocyte cytoskeleton (Williams et al., 2006). From these results we conclude that Basket is necessary, but not sufficient, for stress fiber formation in hemocytes.
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). Cells co-expressing Rac1F37A and Rho1N19 looked very similar to cells expressing Rho1N19 alone (data not shown). This supports the idea that Rac1 is acting through Basket to induce either Rho1 or Rho-kinase activity. It has been reported that Rho GTPase is involved in regulating cytokinesis (Field et al., 1999; Hickson et al., 2006; Piekny et al., 2005), and 42% of hemocytes co-expressing Rac1F37A and Rho1N19 were multinucleate (Fig. 2B,H), indicating a possible cytokinesis defect. Previously, we published that hemocytes expressing dominant negative Rac1 (Rac1N17) were also multinucleate (Fig. 2C,H), had very little F-actin at the cell periphery, and were larger in diameter than controls (Fig. 2C,G) (Williams et al., 2006). Since Rac1 and Rho1 are both know to regulate F-actin formation, this result might be expected if Rho1 activity was inhibited in cells expressing dominant negative Rac1.
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To see if Rho1 could be signaling downstream of Rac1 in hemocytes, we co-expressed constitutively active Rho1 (Rho1V14) with dominant negative Rac1. Co-expression rescued the loss of actin at the cell periphery, the cells were smaller than hemocytes expressing Rac1N17 alone, and there was a partial rescue of the cytokinesis defect (Fig. 2D,F,G). Also, when Rac1N17 or Rho1N19 were overexpressed in hemocytes, the cleavage furrow formed during cytokinesis was never observed. Yet, when dominant negative Rac1 was co-expressed with constitutive active Rho1, some of the cells looked as if they had almost completed cytokinesis (Fig. 2D, arrow). From these results we conclude that Rho1 is indeed signaling downstream of Rac1 in hemocytes.
All of our data thus far point to the idea that Basket is involved in activating the Rho1 pathway in hemocytes. One possibility could be that Basket signaling alleviates some inhibition of Rho1 activity. In resting cells, inactive Rho family GTPases are bound to GDP. Upon signal transduction they are recognized by a guanine nucleotide exchange factor (GEF) and at the same time translocate to a cellular membrane. The GEF promotes the exchange of GDP for GTP leading to activation. Active Rho-family GTPases are then able to interact with their downstream effector proteins. To turn off GTPase signaling active Rho-family GTPases are recognized by a GTPase activating protein (GAP) that accelerates the hydrolysis of GTP to GDP, this shuts-down the Rho-family GTPase (Burridge and Wennerberg, 2004; Ridley, 2001a). One such known inhibitor of Rho1 is the Drosophila GTPase activating protein RhoGAPp190 (Billuart et al., 2001). Expressing a double stranded RNA specific for RhoGAPp190 (RhoGAPp190IR) in hemocytes induced thick stress fiber formation, similar to expression of constitutive active Rho1 (data not shown). When Rac1N17 was co-expressed with RhoGAPp190IR, thin actin fibers were visible in the hemocytes and there was a thick actin ring at the cell periphery (Fig. 2E). This phenotype is very similar to that of hemocytes co-expressing Rac1F37A and dominant negative Rho-kinase, indicating possible Diaphanous activation (Tsuji et al., 2002; Watanabe et al., 1999). The cells were also similar in size to hemocytes co-expressing Rac1N17 and Rho1V14 (Fig. 2F). Finally, only 6% of these cells were multinucleate, all of which had a possible cleavage furrow (Fig. 2G and data not shown). From these results we conclude that activation of Rac1 induces Basket, leading to more Rho1 activity in hemocytes. One possibility is that Basket inhibits RhoGAPp190 activity, though we have not conclusively shown that in this study. Since removing RhoGAPp190 led to a Diaphanous phenotype, and not an activated Rho1 phenotype, we must also consider the possibility that Basket is necessary for Rho-kinase activity.
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; Sinnett-Smith et al., 2001; Tsuji et al., 2002). To examine the possibility that Rho1 feeds back to induce Rac1 in circulating larval hemocytes, we expressed various UAS transgenes expressing Rac1, Rho1 or Rho-kinase. As previously reported (Williams et al., 2006; Zettervall et al., 2004), when wild-type Rac1 was overexpressed in hemocytes it caused release of sessile hemocytes, with a concurrent increase in the number of circulating hemocytes (data not shown and Fig. 3C). No increase in circulating hemocytes was observed when either Rokcatkg or Rho1V14 was overexpressed. Yet, co-expression of Rokcatkg and Rho1V14 in hemocytes induced sessile hemocyte release (Fig. 3A, arrows) and led to a significant increase in the number of circulating plasmatocytes, as well as increased numbers of lamellocytes (Fig. 3C). These phenotypes were blocked by the concurrent expression of dominant negative Rac1 [Fig. 3B arrows (compare Fig. 3A and B, arrows) and 3C]. This suggests that inhibition of Rho-kinase downstream of Rho1 leads to Rac1 activation, as evidenced by sessile hemocyte release, an increase in the number of circulating plasmatocytes, and induction of lamellocyte formation.
To look at hemocytes in more detail we bled larvae and stained the hemocytes with fluorescently labeled phalloidin to visualize their actin cytoskeleton. As has been reported for Rho1 activation, hemocytes expressing constitutive active Rho1 (Rho1V14) had what seemed to be stress fibers running from the center to the periphery of the cell (Fig. 3E) (reviewed by Burridge and Wennerberg, 2004; Ridley, 2001b). When compared to control hemocytes (Fig. 3D), overexpression of Rho1V14 induced a threefold increase in total cellular F-actin (Fig. 3K). Hemocytes overexpressing dominant negative Rho-kinase had a slight increase in F-actin accumulation at the cell periphery, and an approximate twofold increase in total cellular F-actin (Fig. 3F,K). This indicated that blocking Rho-kinase activity alone was not sufficient to induce the Diaphanous-like phenotype. Similar to what was seen in mammalian cells, co-expression of dominant negative Rok and constitutive active Rho1 in hemocytes induced a Rac1 overexpression phenotype. Many hemocytes had ruffled membranes, and there was an eightfold increase in total cellular F-actin (Fig. 3G,K). Many hemocytes co-expressing Rokcatkg and Rho1V14 also extended filopodia (Fig. 3G' and inset). Since Diaphanous signals downstream of Rho1, this could be due to Diaphanous activity (Krebs et al., 2001; Watanabe et al., 1999). Furthermore, in Dictyostelium and mammalian cells it has been shown that Diaphanous-like proteins localize to filopodial tips (Pellegrin and Mellor, 2005; Schirenbeck et al., 2005). When hemocytes co-expressing Rokcatkg and Rho1V14 were stained for Diaphanous expression, it was found to be enriched at the tips of filopodia (Fig. 3G' and inset) similar to what was observed in Dictyostelium and mammalian cells. Concurrent expression of dominant negative Rac1 blocked all of Rokcatkg and Rho1V14 co-expression phenotypes (Fig. 3I,I'). Many of the cells looked similar to hemocytes expressing dominant negative Rho-kinase, with more F-actin at the cell periphery than in control hemocytes and only a twofold increase in total cellular F-actin (Fig. 3I,K). Interestingly, in these cells, Diaphanous was visible at the tips of actin filaments emanating from the center of the hemocytes, but no filopodia were formed (Fig. 3I' and inset, arrows). From these results it is evident that cellular activation, induced by co-expression of constitutive active Rho1 and dominant negative Rho-kinase, requires Rac1 activity.
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). Similar to these results, larval hemocytes co-expressing BskIR and Rho1V14 still had the sessile hemocyte banding pattern, and there was no significant increase in circulating hemocyte numbers (data not shown and Fig. 3C). However, circulating hemocytes co-expressing BskIR and Rho1V14 had a significant increase in total cellular F-actin and were much larger than controls (Fig. 3J,K,L). Also, similar to hemocytes co-expressing Rho1V14 and Rokcatkg, many of the cells had filopodial extensions with Diaphanous enriched at the tips (Fig. 3J' and inset). A similar phenotype was observed when we co-expressed BskIR and RhoGAPp190IR in hemocytes (data not shown). From these results we conclude that both Basket and Rho1 are necessary for the full activation of Rho-kinase.
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). To see if Diaphanous could be signaling between Rho1 and Rac1, we co-expressed dominant negative Rho-kinase (Rokcatkg) and constitutive active Rho1 (Rho1V14) in hemocytes lacking one copy of endogenous diaphanous (dia). Reducing the levels of Diaphanous protein inhibited the hemocyte activation phenotypes observed when Rokcatkg and Rho1V14 were co-expressed. These hemocytes were closer to control cells in diameter, and there were no filopodia extending out from the cell periphery (Fig. 4B). These same cells only had a twofold increase in total cellular F-actin (Fig. 4E). Also, there was no increase in the number of circulating plasmatocytes, as well as no circulating lamellocytes (Fig. 4F). Hemocytes bled from diak07135 heterozygous larvae were similar to wild-type hemocytes in appearance (data not shown). Thus, as in mammalian cells it seems that Diaphanous signals between Rho1 and Rac1 in vivo in Drosophila. Expression of a constitutive active form of Diaphanous (DiaCA) in hemocytes induced sessile hemocyte release, and led to a significant increase in the number of circulating plasmatocytes and lamellocytes (data not shown and Fig. 4F). When the hemocytes were bled, similar to the situation resulting from Rokcatkg and Rho1V14 co-expression, many of the cells expressing DiaCA had filopodia with Diaphanous protein enriched at the tips (Fig. 4D). They also had a significant increase in total cellular F-actin (Fig. 4E). Unlike, Rokcatkg and Rho1V14 co-expression, expression of constitutive active Diaphanous induced extreme membrane ruffling in some hemocytes (Fig. 4C). Co-expression of dominant negative Rac1 could not block the Diaphanous-induced filopodia (Fig. 5A,B), nor could it block the increase in the number of circulating hemocytes (data not shown). Interestingly, coexpression of dominant negative Cdc42 (Cdc42N17) was able to block the Diaphanous filopodia phenotype (Fig. 5A,B). Though similar to Rac1N17, it was unable to block the increase in circulating hemocytes induced by DiaCA (data not shown).
Rac2 is required for filopodia in parasitized larvae
It was shown during Drosophila embryonic development that dominant negative Rac1 actually inhibits both Rac1 and Rac2 activity (Hakeda-Suzuki et al., 2002). This led to the idea that dominant negative Rac1 may also be interfering with the activity of Rac2 in hemocytes. To test this we parasitized larvae using the parasitoid wasp Leptopilina boulardi. In control larvae most of the hemocytes had numerous filopodia extending from the plasma membrane. Similar to hemocytes co-expressing Rokcatkg and Rho1V14, Diaphanous was enriched at the tips of the filopodia (Fig. 6A). Hemocytes from Rac1J11 homozygous mutant larvae also extended numerous filopodia after parasitization. Compared to wild-type hemocytes, the filopodia appeared to be thinner and possibly had less Diaphanous protein at the tips. Hemocytes from Rac2 homozygous mutants did not extend filopodia. Diaphanous could be seen at the end of actin structures extending from the center of the cell, but these never extended passed the cell periphery (Fig. 6A, arrowhead). From these data we conclude that after parasitization, Rac2, and not Rac1, is necessary for filopodia formation. This also means that similar to what was observed during embryonic development, dominant negative Rac1 may interfere with Rac2 activity in hemocytes (Hakeda-Suzuki et al., 2002).
While doing these experiments we noticed that both Rac1J11 and Rac2 mutations had an effect on the formation of lamellipodia in lamellocytes. After parasitization some lamellocytes in control larvae had extremely ruffled membranes (Fig. 6B). These ruffles were actin rich, but contained very little Diaphanous protein within the ruffles. Instead, Diaphanous was enriched at the base of the ruffling. Lamellocytes from both Rac1J11 and Rac2 mutants had very little membrane ruffling after parasitization, and Diaphanous expression was not localized (Fig. 6B). From these results we conclude that both Rac1 and Rac2 are required for lamellipodia formation in activated lamellocytes.
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). Basket may inhibit RhoGAPp190, inducing more Rho1 activation. Together Rho1 and Basket activate Rho-kinase. Activation of Rho-kinase inhibits Diaphanous-induced Rac1 activity, leading to thick stress fiber formation and disruption of cellular adhesions, allowing the hemocytes to circulate freely. Blocking the ability of Diaphanous to induce Rac1 would eventually lead to less Rho1 activity, thus stabilizing cellular adhesions via Diaphanous, which might help the hemocytes to adhere to the wasp egg. This inter-regulation between Rac1 and Rho1 would then allow a cell to regulate its migration and/or adhesion properties.
Inter-regulation of Rac1 and Rho1
In mammalian cell lines it has been shown that the inhibition of Rho-kinase downstream of Rho GTPase proteins can lead to the induction of Rac activity (Salhia et al., 2005; Sinnett-Smith et al., 2001; Tsuji et al., 2002). It has also been well documented that Rac can induce Rho activation (reviewed by Ridley, 2001a; Burridge and Wennerberg, 2004; Ridley, 2001b). What has not been shown is exactly how Rac can activate the Rho pathway. Our data suggests that the Drosophila c-Jun N-terminal kinase, Basket, is necessary for Rho1- and Rho-kinase-induced stress fiber formation. Jun kinase has been shown to be a signaling component downstream of Rho signaling or stress fiber formation, or as a regulator of transcription of the building blocks needed for actin formation (Xia and Karin, 2004). However, our data is the first evidence that Jun kinase may have a more direct role in stress fiber formation.
The overexpression of Rac1F37A in hemocytes induces thick actin stress fibers, and this requires the activity of Rho-kinase. Yet when Basket was removed downstream of Rac1F37A no stress fibers were formed. This could mean that Basket is necessary for the activity of the Rho1 pathway, or alternatively it could mean the Basket is more directly involved in stress fiber formation. One possibility is that Basket is inhibiting the Rho1 inhibitor RhoGAPp190. Billuart et al. (Billuart et al., 2001) showed in Drosophila that maturing neurons require RhoGAPp190 activity to inhibit Rho1. It was also observed that overexpression of RhoGAPp190 was sufficient to inhibit Rho1 activity during Drosophila wing development (Chen et al., 2005). Here we show that lowering RhoGAPp190 activity in hemocytes is sufficient to induce a Rho1 activation phenotype. Yet when RhoGAPp190 is removed in cells lacking Basket activity, instead of a Rho1 phenotype, the cells have a phenotype reminiscent of Diaphanous activity. If lowering the expression of RhoGAPp190 was sufficient to fully activate the Rho1 pathway, including Rho-kinase, then the cells should look similar to cells expressing constitutive active Rho1. This leaves us with two possibilities: Rho-kinase and Jun kinase are both required for actin bundling leading to thick stress fiber formation, or alternatively Basket is regulating the activity of Rho-kinase. Previously, and again in this paper, it has been shown that inhibiting Rho-kinase downstream of Rho GTPase induces Rac activity. If Basket is necessary for Rho-kinase activation, then removing Basket in a cell where Rho1 is activated should induce a Rac1-like phenotype. This is exactly what is seen when Basket is removed in hemocytes expressing constitutive active Rho1. There was very little difference in the cellular phenotypes observed between removal of Basket and inhibition of Rho-kinase in hemocytes expressing constitutive Rho1. From these results we believe that Basket is somehow necessary for Rho-kinase activation. There is still the possibility that inhibiting stress fiber formation in cells where Rho1 is active leads to Rac1 activation, and this should be tested in future.
Rac2 required for filopodia formation
We have shown previously that parasitization also induces filopodia on larval hemocytes (Williams et al., 2005). Here we show that inhibiting Rho-kinase downstream of activated Rho1, or overexpression of constitutive active Diaphanous, both led to filopodia expression in hemocytes. Interestingly, co-expression of dominant negative Rac1 could block filopodia induced by co-expression of dominant negative Rho-kinase and constitutive active Rho1, yet was unable to inhibit filopodia induced by constitutive active Diaphanous. This would mean that dominant negative Rac1 was able to block the activity of endogenous Diaphanous, but unable to inhibit constitutive active Diaphanous. Constitutive active Diaphanous was made by removing the Rho-binding N terminus, as well as the auto-inhibitory domain at the C terminus (Olson, 2003; Somogyi and Rorth, 2004). Furthermore, similar to the diaphanous mammalian homologues, Drf2 and Drf3, Drosophila Diaphanous contains a putative Cdc42 and Rac interactive binding (CRIB) domain near the N terminus (see supplementary material Fig. S1) (Peng et al., 2003). It is possible that since the constitutive active Diaphanous allele lacks this domain, as well as auto-inhibitory domains, Rac is no longer able to regulate Diaphanous activity. Also, we show that Cdc42 is able to activate the cellular immune response and interacts genetically with Diaphanous. Thus, there is the possibility that endogenous Diaphanous signals to activate Rac GTPase (either Rac1, Rac2 or both), and together Rac and Diaphanous activate Cdc42. Constitutive active Diaphanous may bypass the need for Rac GTPase, and still activate Cdc42.
Interestingly, cells lacking Rac2 activity or expressing dominant negative Rac1 look as if they begin the process of making a filopodia, but cannot extend this process past the cell periphery. It has been well documented that Diaphanous-like formins are involved in making filopodia (Pellegrin and Mellor, 2005; Peng et al., 2003; Schirenbeck et al., 2005). Thus far, Diaphanous-like formins have been shown to interact with either Cdc42 or Rif GTPase during filopodia formation (Peng et al., 2003). Although we have yet to show a direct interaction, here we show the possibility, that in hemocytes, Diaphanous may require the activity of Rac2 for filopodia extension. In Dictyostelium and mammalian cells Diaphanous-like proteins were seen at the end of filopodia, and the same is true for hemocytes. In Rac2 or dominant negative Rac1 mutants, Diaphanous was observed at the end of a growing actin filament, but this never extended past the cell periphery. It seems that the signal activating Diaphanous to induce filopodia formation is still intact in Rac2 mutants, but Diaphanous requires the activity of Rac2 to continue the process past the cell periphery. We have also observed that Diaphanous requires the activity of Cdc42 for filopodia formation, and that unlike dominant negative Rac1, Cdc42 is able to inhibit filopodia formation induced by constitutive active Diaphanous. We speculate that Rac2 is signaling upstream of Diaphanous for filopodia formation, and Cdc42 is signaling downstream.
Concluding remark
Finally, though there is strong evidence for reciprocal regulation in mammalian cells, we must caution that some of the phenotypes achieved by overexpression of the various alleles used in this study may be due to artifacts. In the past, experiments expressing dominant negative Rho family constructs have given strong embryonic phenotypes. Yet, more recent studies using loss-of-function alleles of the same genes have failed to reproduce the overexpression phenotypes. More studies using mutated endogenous alleles will be needed to confirm the phenotypes observed in this study.
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), Hemolectin-Gal4, 2X UAS-eGFP was provided by Sergey Sinenko (Sinenko et al., 2004), UAS-DiaCA was provided by Pernille Rørth (Somogyi and Rorth, 2004). Flies were kept on a standard mash-potato diet at between 21-25°C. Stocks crossed to Gal4 driver flies, and the uncrossed control flies, were raised at 29°C. The G486 strain of Leptopilina boulardi (Dupas et al., 1998) 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 agar plates.
Immunofluorescence
For all antibody stainings, hemocytes were bled from larvae into 20 µl of phosphate-buffered saline (PBS), and allowed to attach to a glass slide (SM-011, Hendley-Essex, Essex, UK) for 1 hour. Staining and analysis were done according to Williams et al. (Williams et al., 2006). The polyclonal anti-Diaphanous (Afshar et al., 2000) was diluted 1:2,500 in 3% BSA/PBS. Alexa Fluor-phalloidin (Molecular Probes) was diluted to a final concentration of 0.20 µg/µl in 1x PBS. Cell were visualized using epifluorescence and digital images were obtained with a Hamamatsu C4742-95 video unit, controlled by the Openlab program (Improvision, Coventry, UK), or a Zeiss Axiophot microscope equipped with a KAPPA DX20HC CCD camera. Photoshop (version 7.0, Adobe Systems, San Jose, CA, USA) and Imagetrak (created by Peter K. Stys, Ottawa Health Research Institute, Ottawa, Canada) were used for digital editing. Imagetrak was used to measure fluorescent intensity.
To visualize hemocyte patterns within larvae, wandering third instar larvae were washed in PBS, and then killed by freezing at -80°C for 1.5 minutes. The larvae were then transferred to a glass slide and covered in 50% glycerol and visualized as described previously.
Hemocyte counting and statistics
Hemocyte counting and statistics were done as described previously (Zettervall et al., 2004). Briefly, UAS transgenic lines were crossed to Hemolectin-Gal4, 2X UAS-eGFP. The females were allowed to lay eggs at 21-25°C for 2 days before the vials were moved to 29°C. Larvae were staged according to procedures described by Andres and Thummel (Andres and Thummel, 1994). Staged larvae were washed in PBS before being bleed into 20 µl of PBS, by using a fine pair of forceps and a 27-gauge needle. The PBS containing the hemocytes was then loaded onto a Neubauer improved hemocytometer for counting. Hemocytes from at least 15 larvae from each strain were counted, and statistical analysis was done according to the procedures outlined by Zettervall et al. (Zettervall et al., 2004).
Measurement of cell size
Cells were measured according to the method of Williams et al. (Williams et al., 2006). Cell area was calculated from measurements of the X and Y axes using the Openlab program, and taking the average of these two measurements in microns. For statistics. an initial ANOVA indicated that the overexpression of the UAS constructs significantly affect hemocyte cell size. Multiple t-tests were performed to study specific interactions between certain genotypes and their corresponding crosses. ANOVA analysis was performed using a program available at http://www.physics.csbsju.edu/stats/anova.html. Microsoft Excel and Graphpad (http://www.graphpad.com/quickcalcs/ttest1.cfm) were used for t-test analysis.
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Acknowledgments |
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-Gal4, 2X UAS-eGFP flies, Pernille Rørth for the UAS-DiaCA flies. This research was supported by grants from the Swedish Research Council, the Swedish Cancer Society and the Wallenberg Consortium North. |
Footnotes |
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References |
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