Drosophila CYRI controls lamellipodial protrusions. (A) Insertion of a GFP-tag at the 3′ end of the cyri gene in S2 cells using CRISPR/Cas9-mediated genome editing. Lysates from control cells and cells expressing CYRI-GFP were probed on a Western blot using an anti-CYRI (left) or anti-GFP (right) antibody. (B) Quantification of spread cell area, Cas9 S2 control: n = 200 cells; S2 CYRI-GFP knock-in: n = 200 cells; To evaluate statistical significance, the Mann–Whitney test was used and the following P value (two- tailed) was obtained: P value: ns < 0.05. (C–C″) Confocal images of CRISPR/Cas9-edited S2 cells expressing CYRI-GFP (C endogenous GFP; green) stained with phalloidin-Alexa568 (C′ red) and DAPI (C″ blue). Scale bars represent 10 µm. (D and E) Time-lapse fluorescence microscopy images of S2 CYRI-GFP knock-in cells. Images were taken at indicated timepoints. Black arrowheads mark lamellipodial protrusions in D and macropinocytic structures in E enriched for endogenous CYRI-GFP. Scale bar represents 10 µm. (F–H) Confocal images of S2R+ cells stained with phalloidin-Alexa488 (grey) and an anti-CYRI antibody (magenta) transfected with (F) an EGFP, (G) a wild type CYRI (CYRI^WT) or (H) a mutant CYRI-^R163/164D construct. (I) Quantification of cells showing a spiky cell morphology. n = 100 cells for each genotype from three independent transfection experiments. Two-sided Fisher’s exact test was used. P value: *** P < 0.0001; ns: > 0.05. Source data are available for this figure: SourceData F2.