The Arp2/3 complex is required for ARS formation and for maintenance of BBB function. (A–C) Nerve cord of third instar larvae expressing Arp3-GFP fusion protein driven by Moody-Gal4. The Arp3-GFP (A) colocalizes with the ARSs labeled with phalloidin (B). The merged image is shown in C. Arrowheads in A–C indicate ARSs colabeled with phalloidin and Arp3-GFP. (D–F) Nerve cord from larvae expressing RNAi for both Arp2 and Arp3 proteins labeled for phalloidin (D). The larvae also carried the Nrg-GFP protein trap. Nrg-GFP is shown in E, and the merged image is shown in F. Arrows show aberrant ARSs that no longer associate with Nrg-GFP labeling. Arrowheads indicate sites lacking Nrg-GFP continuity. (G) Dye penetration to the nerve cord was measured by the fluorescent intensity of nerve cords dissected from wild-type (WT) larvae and compared with moody mutant larvae (moody^Δ17). The difference between the averaged fluorescent intensity of the two groups was calculated by Student’s t test and was found to be significant (***, P < 0.0066). (H) Dye penetration was compared between three groups: control larvae carrying RNAi to the Arp2/3 components alone (UAS-sop2i;arp3i/+), control larvae carrying moody-gal4 alone (moody-gal4/+), or an experimental group of Arp2/3 knockdown larvae (moody-gal4:UAS-sop2i;arp3i). One-way ANOVA test with Dunnett’s test (using SAS program) was used to determine the statistical significance of the difference between the experimental and the control groups (Moody-gal4/+ or Sop2i;Arp3i/+). In both cases, the difference between the experimental group and each of the control groups was statistically significant (at α = 0.05; indicated by asterisks). (G and H) Error bars indicate standard deviation.