Drosophila CG32066 is the ortholog of human CYRI. (A) Sequence alignment of human CYRI-A, CYRI-B and Drosophila CG32066. Conserved amino acid residues are marked by asterisks. Colons indicate conserved positions containing residues with strongly similar properties. Gaps are indicated by dashes. The conserved two arginine residues, R163 and R164 are highlighted in red. The highest conservation is found within the DUF1394 domain (highlighted in yellow). (A′) The Drosophila CYRI protein shares 52.52% identity with the human (Hs) CYRI-B protein and 55.17% identity with the human (Hs) CYRI-A protein. The human proteins share 79.57% identity. (B) Western blot from pull-down of GST control, GST-Rac1WT, or GST-Rac1Q61L beads, with cell lysate expressing either HA-tagged wild type CYRI or HA-tagged mutant CYRI-R163/164D variant. (B′) Quantification of (B) from three independent experiments. Signals were normalized to loaded input (1% of the starting lysate material). Mean ± SD. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons. * P < 0.05. (C and D) Confocal images of macrophages isolated from (C) wild type and (D) trans-heterozygous cyri mutant pupae (cyriΔ11/Df[ED4457]), stained with phalloidin-Alexa488 (grey), DAPI (blue), and an anti-CYRI antibody (green). Note: no differences in immunofluorescent anti-CYRI intensity were detected. (E–H) Confocal images of (E) wild type and (F) homozygous cyriΔ2 mutant (G) transheterozygous cyriΔ2/Df(ED4457) mutant and (H) transheterozygous cyriΔ11/Df(ED4457) mutant macrophages were co-stained with phalloidin (grey) and DAPI (blue). High magnification of boxed areas displays single cells. Scale bars represent 10 µm. (I) Quantification of spread cell area, n = 167 for all genotypes. To evaluate statistical significance, one-way-ANOVA (Kruskal–Wallis test with Dunn´s correction) was used. P value: <0.001 (***). The red bar represents the median. Three independent experiments for each genotype were performed. Source data are available for this figure: SourceData FS1.