Tws function is required in the cytoplasm. (A) Tws-GFP variants constructed for transgenic expression. Tws-GFP was fused to two copies of NLS^SV40 or to a mutated form (NLSm^SV40; substitutions in red). (B) The localization of Tws variants fused to GFP was visualized in third instar larval tissues. Salivary gland cells are shown (see also brain cells in Fig. S1). Transgenes were under the UASp promoter, and expression was driven by Ubi-Gal4. Merge images show GFP (green) and DNA stained with Hoechst 33342 (blue). Scale bar: 20 µm. (C) The nuclear/cytoplasmic ratio of GFP intensity was quantified (mean ± SD). (D) Genetic rescue of tws^aar1/tws^P mutant flies by the expression of the indicated Tws variants. Values shown correspond to percentages of eclosed tws^aar1/tws^P pupae (Tb⁺) relative to the expected number of tws^aar1/tws^P pupae calculated from the total number of eclosed pupae. See Materials and methods for details. Values are averages of three independent experiments in which between 110 and 1252 eclosed pupae were scored for each cross. Error bars: SD. ****, P ≤ 0.0001.