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First published online 18 April 2006
doi: 10.1242/jcs.02920

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Rac1 signalling in the Drosophila larval cellular immune response

Umeå Centre for Molecular Pathogenesis (UCMP), Umeå University, S-901 87, Umeå, Sweden

View larger version (39K): [in a new window]   Fig. 1. Rac1 effector loop mutants fail to disrupt the sessile hemocyte population. (A-F) GFP expression in sessile hemocytes of control larvae and larvae expressing various alleles of Rac1 (A) He-Gal4 (B) UAS-Rac1; He-Gal4 (C) UAS-Rac1F37A; He-Gal4 (D) UAS-Rac1Y40C; He-Gal4 (E) UAS-Rac1F37A; UAS-Rac1Y40C; He-Gal4 (F) UAS-Rac1N17/He-Gal4. (G) Hemocyte counts after overexpression of various Rac1 alleles. He-Gal4 was crossed with the different Rac1 alleles. Hemocytes were counted from at least 15 individual larvae. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains.

View larger version (25K): [in a new window]   Fig. 2. Rac1 must activate two pathways to induce hemocytes activation. (A-F) Hemocyte actin cytoskeleton was visualized using TRITC-phalloidin (red), the nucleus was stained with DAPI (blue) (A) He-Gal4 (B) UAS-Rac1; He-Gal4 (C) UAS-Rac1F37A; He-Gal4, hemocytes were also stained with anti-phosphorylated-tyrosine antibody (green) (D) UAS-Rac1Y40C; He-Gal4 (E) UAS-Rac1F37A; UAS-Rac1Y40C; He-Gal4 (F) UAS-Rac1N17/He-Gal4. (G) F-actin expression levels of the various Rac1 alleles. He-Gal4 was crossed with different Rac1 alleles and hemocytes were bled from wandering third-instar larvae. The hemocytes were stained with TRITC-phalloidin. Imagetrak was used to measure fluorescence intensity of at least 100 hemocytes from three different larvae. Different letters indicate similar groups (i.e. `a' is significantly different from `b' or `c' and so on; Student's t-test, P<0.01). (H) Determination of plasmatocyte diameter. The cell diameter of plasmatocytes from the various genotypes was measured, as described in Materials and Methods, and the diameter (µm) for 25 hemocytes was plotted. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains.

View larger version (28K): [in a new window]   Fig. 3. Reducing the amounts of cofilin partially rescues Rac1Y40C. (A-E) Hemocytes were bled from early third-instar larvae and stained with TRITC-phalloidin (red), the nucleus was stained with DAPI (blue). (A) UAS-Rac1;He-Gal4 (B) UAS-Rac1Y40C; He-Gal4 (C) y[1] w[67c23]; P{w[+mC]=lacW}tsr[k05633]/CyO, Kr-Gal4, UAS-GFP (D) UAS-Rac1Y40C/P{w[+mC]=lacW}tsr[k05633]; He-Gal4 (E) He-Gal4. (F) F-actin expression levels of different Rac1 alleles crossed to He-Gal4 and y[1] w[67c23]; P{w[+mC]=lacW}tsr[k05633]/CyO, Kr-Gal4, UAS-GFP. Hemocytes were bled from wandering third-instar larvae and stained with TRITC-phalloidin. Imagetrak was used to measure fluorescence intensity of at least 100 hemocytes from three different larvae. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains. (G) Hemocyte counts after overexpression of various UAS alleles. Hemocytes were counted from at least 15 individual larvae. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains. (H) Determination of plasmatocyte size. The cell diameter of plasmatocytes from the various genotypes was measured, as described in Materials and Methods, and the diameter (µm) for 25 hemocytes was plotted.

View larger version (29K): [in a new window]   Fig. 4. Bsk activation in hemocytes. Hemocytes were recovered from early third-instar larvae and stained for Bsk activation using anti-phosphorylated-JNK antibody (red). Cells were counter-stained with FITC-phalloidin to visualize their actin cytoskeleton (green). In the merged pictures, overlap of the two stainings is yellow. (A) He-Gal4 (B) UAS-BskA-Y; He-Gal4 (C) UAS-Rac1; He-Gal4 (D) UAS-Rac1Y40C; He-Gal4 (E) UAS-Rac1F37A; He-Gal4. (F) Quantification of Bsk activity. Imagetrak was used to measure fluorescence intensity of at least 100 hemocytes from three different larvae. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains.

View larger version (25K): [in a new window]   Fig. 5. Bsk is necessary for Rac1-induced increases in circulating hemocytes. (A,C,E) GFP expression in sessile hemocytes larvae expressing (A) UAS-Rac1; He-Gal4 (C) UAS-BskIR/He-Gal4 (E) UAS-Rac1;UAS-BskIR/He-Gal4. (B,D,F) In respective larvae, hemocyte actin cytoskeleton was visualized with TRITC-phalloidin, the nucleus was stained with DAPI. Arrows indicate cells not expressing the transgene. The He-Gal4 driver flies also contain an UAS-GFPnls transgene. GFP was used to indicate transgene expression. GFP expression is not shown in these figures. (G) Hemocyte counts after overexpression of various UAS alleles. Hemocytes were counted from at least 15 individual larvae. (H) Determination of plasmatocyte diameter. The cell diameter of plasmatocytes from the various genotypes was measured on their x and y axes, the average of 25 hemocytes was plotted in µm. *, significant difference (Student's t-test, P<0.01) compared with the parental UAS and He-Gal4 strains.

View larger version (35K): [in a new window]   Fig. 6. Loss of Rac1 or Bsk does not block lamellocyte activation after parasitization. Hemocytes were recovered from larvae 40 hours after parasitization by the parasitoid L. boulardi G486. (A) Lamellocytes were recovered from parasitized Hml-Gal4 control, UAS-BskIR/Hml-Gal4 or, homogygous Rac1J11 loss-of-function larvae and stained with the L1 lamellocyte-specific antibody (green), the nucleus was stained with DAPI (blue). (B) Lamellocytes recovered from parasitized Hml-Gal4 control, UAS-BskIR/Hml-Gal4, or homogygous Rac1J11 loss-of-function larvae and stained with anti-phosphorylated-FAK antibody (red), anti-phosphorylated-tyrosine antibody (green) and Alexa-Fluor-350-phalloidin (blue). In the merged pictures, overlap of the three stainings is light violet (arrows). (C) Encapsulation of wasp eggs in parasitized Hml-Gal4 control UAS-BskIR/Hml-Gal4 larvae or homogygous Rac1J11 loss-of-function larvae. Numerical values for proper encapsulation percentages [(Number of properly melanized wasp eggs/number of parasitized larvae) x 100] are presented above each bar. Numbers indicate the number of wasp-parasitized larvae. Numbers in parentheses indicate percentage of total larvae with a properly melanized wasp egg.

View larger version (24K): [in a new window]   Fig. 7. Bsk activation in parasitized hemocytes. (A) Hemocytes were recovered from non-parasitized and parasitized control (Rac1J11/TM6b,Tb) or homogygous Rac1J11 loss-of-function third-instar larvae and stained for Bsk activation with anti-phosphorylated-JNK antibody (red), anti-phosphorylated-tyrosine antibody (green), and Alexa-Fluor-350-phalloidin (blue). (B) Quantifying Bsk activity. Imagetrak was used to measure fluorescence intensity of at least 100 hemocytes from three different larvae. Different letters indicate similar groups (i.e. `a' is significantly different from `b' or `c' and so on; Student's t-test, P<0.01).

View larger version (25K): [in a new window]   Fig. 8. Schematic diagram showing Rac1 involvement in lamellipodia formation.