npg Fused dimerization transduces Hedgehog signal 1442 Figure 3 The dimerization of Fu is induced by Hh and is Smo and Cos2 dependent. (A, B) FRET efficiency of Fu-CFPN and Fu-YFPN in S2 cells (mean ± sd, n ≥ 15) (A), or in wing discs (mean ± sd, n ≥ 10) (B). (C, D) In the absence/presence of Hh, FRET efficiency of Fu-CFPN and Fu-YFPN co-expressing Smo (C) or Cos2 (D) with control RNAi, Cos2 RNAi (C) or Smo RNAi (D), to knockdown the endogenous protein. (E) Smo-CFPC, Cos2-YFPC or Cos2ΔMD-YFPC and Myc-Fu were co- transfected in S2 cells, with or without Hh treatment, to detect the effects of the Cos2 motor domain on the co-localization of Smo and the Cos/Fu complex. Myc-Fu was immunostained (blue). (E′) Quantification of Smo that co-localized with Cos2 or Cos2ΔMD in the absence or presence of Hh. (F) FRET efficiency of Smo-CFPC and Cos2-YFPC or Cos2ΔMD-YFPC with Myc- Fu co-transfected in S2 cells with or without Hh treatment. (G) FRET efficiency of Smo-CFPC and Cos2-YFPC with control RNAi, or the endogenous Fu knocked down by Fu RNAi in the absence/presence of Hh, respectively. mutant in which Gly 13 is replaced with Val [15, 27]. Fu kinase (FuK) only contains the Fu kinase domain (amino acids 1-306 of Fu), while FuK* carries both the Fu ki- nase domain and the Su(fu) binding region (amino acids 1-436) [7]. We first used the ptc-luciferase assay, a tran- scriptional reporter for Hh activity, to test the pathway activation by various Fu dimers. We found that either Fv2-Fu treated by AP20187 or Div-Fu was able to acti- vate ptc gene expression (Figure 4A-4B). The dimerized FuK domain had similar activity to full-length Fu, while dimerized FuK* induced the highest reporter activity, which might be due to its phosphorylation on Su(fu) (Figure 4A; see below). FKBP-based dimerization system has a unique ad- vantage. The different concentrations of AP20187 can induce Fu dimerization at different levels and may mimic Hh gradient. Following transfection with Fv2-Fu and treatment with increased concentration of AP20187 in S2 cells, ptc luciferase reporter activity was induced in a graded manner, suggesting that graded Fu dimerization may activate the Hh pathway to varying amounts (Figure 4C). As Smo may transduce a graded Hh signal across Cell Research | Vol 21 No 10 | October 2011