Drosophila CYRI binds activated Rac1 and controls lamellipodial protrusions. (A–A″) Comparisons of the highly similar structures and topologies of (A) CG32066 with human (A′) CYRI-A and (A″) CYRI-B based on AlphaFold2 protein structure predictions (Jumper et al., 2021). As recently shown by crystal structure analysis CYRI proteins comprised solely of α-helices (Kaplan et al., 2020; Yelland et al., 2021). Note the model contains the two highly conserved arginines at positions 163 and 164 (marked in yellow) in the fly protein corresponding to R161 and R162 in human CYRI-B. (B) Sequence alignment of Drosophila CG32066, human CYRI-A, and CYRI-B shows conservation of the arginines at positions 163 and 164. (C) Comparative sequence analysis between CG32066 and human CYRI-A and CYRI-B proteins. The numbers refer to Clustal W sequence alignment score (Thompson et al., 1994). (D) Pull-down experiments with GST-CYRI proteins. GSH-sepharose-bound GST-CYRI (wild type or R163/164D mutant) were preloaded with GDP or GTPγS and incubated with lysate from S2R+ cells transfected with either constitutively activated Rac1-V12 or dominant-negative Rac1-N17 construct. (E) Quantification of (D) from three independent experiments. Signals were normalized to GST. Mean ± SD. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons. * P <0.05. (F) Visualization of CYRI and Rac1 BIFC interaction in wing imaginal discs. Maximum intensity projection images of wing imaginal discs expressing the indicated Split-YFP construct combinations in the en-Gal4 pattern. Expression of transgenes is verified by antibody staining as indicated. Anterior is to the left. (G) Co-expression of Rac1-myc-NYFP and wild type CYRI-HA-CYFP leads to reconstitution of YFP, whereas (G) co-expression of Rac1-myc-NYFP and mutant CYRI-^R163/164D-HA-CYFP does not show YFP fluorescence. Three independent experiments for each genotype were performed. Scale bars represent 50 µm. Source data are available for this figure: SourceData F1.