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First published online 16 January 2007
doi: 10.1242/jcs.03341

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Reciprocal regulation of Rac1 and Rho1 in Drosophila circulating immune surveillance cells

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

View larger version (20K): [in this window] [in a new window]   Fig. 1. Rac1 activates Basket to induce Rho-kinase activity (A-D) Hemocyte actin cytoskeleton was visualized using Alexa Fluor 546-phalloidin. (A) Hml-Gal4 controls, (B) UAS-Rac1F37A;Hml-Gal4, (C) UAS-Rac1F37A;UAS-Rokcatkg/Hml-Gal4,(D) UAS-Rac1F37A;Hml-Gal4;UAS-BskIR. (E) Stress fiber phenotypes. The hemocytes from five different larvae were counted to determine the percentage of cells having either thick or thin stress fibers [(number of hemocytes with phenotype/total number of hemocytes) x 100]. An asterisk indicates a significant difference (Student's t-test, P<0.01) compared with the parental UAS and Hml-Gal4 strains.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Rac1, via Basket, induces Rho1 activity. (A-E) The hemocyte actin cytoskeleton was visualized using Alexa Fluor 546-phalloidin (red) and the nucleus was stained with DAPI (blue). (A) Hml-Gal4 controls. (B) UAS-Rac1F37A;Hml-Gal4;UAS-Rho1N19. (C) Hml-Gal4;UAS-Rac1N17.(D) Hml-Gal4;UAS-Rac1N17/UAS-Rho1V14; arrow indicates possible cytokinesis cleavage furrow. (E) Hml-Gal4;UAS-Rac1N17/UAS-RhoGAPp190rnai. (F) 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. Different letters indicate similar groups (i.e. `a' is significantly different than `b' or `c' and so on. Student's t-test, P<0.01). (G) Percentage of multinucleate cells. The hemocytes from five different larvae were counted to determine the percentage of multinucleate cells as determined by DAPI staining [(number of multinucleate hemocytes/total number of hemocytes) x 100]. An asterisk indicates a significant difference (Student's t-test, P<0.01) compared with the parental UAS and Hml-Gal4 strains

View larger version (40K): [in this window] [in a new window]   Fig. 3. Activating Rho1 while inhibiting Rho-kinase activates Rac1. (A,B) GFP expression in sessile hemocytes. (A) UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14, (B) UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14/UAS-Rac1N17. Arrows indicate location of sessile hemocyte population. (C) Hemocyte counts after overexpression of various UAS alleles. Hml-Gal4 was crossed with the different UAS constructs. Hemocytes were counted from at least 15 individual larvae. An asterisk indicates a significant difference (Student's t-test, P<0.01) compared with the parental UAS and Hml-Gal4 strains. (D-J) The hemocyte actin cytoskeleton was visualized using Alexa Fluor 546-phalloidin (red) and the nucleus was stained with DAPI (blue). (D) Hml-Gal4,(E) Hml-Gal4;UAS-Rho1V14,(F) UAS-Rokcatkg/Hml-Gal4,(G) UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14, (G') UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14 [anti-Diaphanous (red), Alexa Fluor 488-phalloidin (green)], (H) UAS-Rac1;Hml-Gal4, (I) UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14/UAS-Rac1N17,(I') UAS-Rokcatkg/Hml-Gal4;UAS-Rho1V14/UAS-Rac1N17 [anti-Diaphanous (red), Alexa Fluor 488-phalloidin (green)]. Arrows indicate Diaphanous localization at the tips of an actin structure. (J) Hml-Gal4;UAS-Rho1V14/UAS-BskIR,(J') Hml-Gal4;UAS-Rho1V14/UAS-BskIR [anti-Diaphanous (red), Alexa Fluor 488-phalloidin (green)]. (K) F-actin expression levels. Hml-Gal4 was crossed with different UAS constructs and hemocytes were bled from wandering third instar larvae. The hemocytes were stained with Alexa Fluor 546-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 than `b' or `c' and so on. Student's t-test, P<0.01). (L) 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. Different letters indicate similar groups (i.e. `a' is significantly different than `b' or `c' and so on. Student's t-test, P<0.01).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Rho1 signals through Diaphanous to activate Rac1. (A-D) The hemocyte actin cytoskeleton was visualized using Alexa Fluor 546-phalloidin (red) and the nucleus was stained with DAPI (blue). (A) Hml-Gal4, (B) diak07135/UAS-Rokcatkg;UAS-Rho1V14/Hml-Gal4, (C) Hml-Gal4/DiaCA,(D) Hml-Gal4/DiaCA [anti-Diaphanous (red), Alexa Fluor 488-phalloidin (green)]. (E) F-actin expression levels. Hml-Gal4 was crossed with different UAS constructs and hemocytes were bled from wandering third instar larvae. The hemocytes were stained with Alexa Fluor 546-phalloidin. Imagetrak was used to measure fluorescence intensity of at least 100 hemocytes from three different larvae. An asterisk indicates a significant difference (Student's t-test, P<0.01). (F) Hemocyte cell counts. Hemocytes were counted from at least 15 individual larvae. An asterisk indicates a significant difference (Student's t-test, P<0.01) compared with the Hml-Gal4 strain.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Dominant negative Cdc42 can block Diaphanous-induced filopodia. (A) Plasmatocytes recovered from Hml-Gal4;DiaCA/Rac1N17 or Hml-Gal4/Cdc42N17;DiaCA larvae and stained with anti-Diaphanous (red) and Alexa Fluor 488-phalloidin (green). In the merged images, overlap of expression appears yellow. (B) Cell counts of hemocytes expressing multiple filopodia. Hemocytes were counted from at least five individual larvae. An asterisk indicates a significant difference (t-test, P<0.01) compared with the Hml-Gal4 strain.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Rac GTPases are necessary for cell extensions after parasitization. (A) Rac2 is necessary for filopodia. Plasmatocytes recovered from parasitized w1118 control, homozygous Rac1J11 or Rac2 loss-of-function larvae and stained with anti-Diaphanous (red), and Alexa Fluor 488-phalloidin (green). In the merged images, overlap of expression appears yellow. Arrowhead indicates Diaphanous at the tip of an actin structure. (B) Rac1 and Rac2 are necessary for lamellipodia. Lamellocytes recovered from parasitized w1118 control, homozygous Rac1J11 or Rac2 loss-of-function larvae and stained with anti-Diaphanous (red) and Alexa Fluor 488-phalloidin (green). In the merged images, overlap of expression appears yellow.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Schematic diagram showing reciprocal regulation of Rac1 and Rho1 in hemocyte activation.