Fig. 5: Asp33, Asp75, and Glu117 are required for the phosphatase activity of cathD.

a, b Representative Western blots indicating p-cofilin levels in extracted proteins from adult flies. Expression of wild-type cathD fully rescues the elevated p-cofilin levels caused by cathD depletion (cathD¹; da > cathD^wt), whereas expression of mutant cathD carrying Asp33 (cathD¹; da > cathD^D33G), Asp75 (cathD¹; da > cathD^D75G), or Glu75 (cathD¹; da > cathD^E117G) in cathD¹ background does not restore the elevated p-cofilin levels, suggesting that mutating any of these three residues abolishes cathD phosphatase activity. Quantifications of p-cofilin/cofilin ratio (normalized to w¹¹¹⁸) are shown in b. Note that p-cofilin levels remain elevated upon expressing cathD mutants in cathD¹ background (cathD¹; da > cathD^D33G, cathD¹; da > cathD^D75G and cathD¹; da > cathD^E117G). c Actin-based bristle structures in pupae carrying different genotypes. Ubiquitous expression of the wild-type (cathD¹; da > cathD^wt) or a proteolytically inactive mutant of cathD (cathD¹; da > cathD^D231N) restores the over-protruded bristle phenotype upon cathD depletion (cathD¹) at 34 h APF. No significant effects were detected upon expressing cathD^D33G (cathD¹; da > cathD^D33G), cathD^D75G (cathD¹; da > cathD^D75G) or cathD^E117G (cathD¹; da > cathD^E117G) and bristles remain defective. Bristles were labeled by phalloidin. Dash-boxed regions are enlarged in lower panels. d The ratio of individual flies with disorganized actin bundles in bristles, showing that ubiquitous expression of cathD^wt or a proteolytic inactive cathD^D231N reduces the defective ratio, whereas expression of cathD^D33G, cathD^D75G, or cathD^E117G exhibits minimal effect. Scale bar, 5 mm. Data are means ± SEM. One-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001.