npg Fused dimerization transduces Hedgehog signal 1438 far away from the N- or C-terminus of Fu. This may be the reason why we could not observe a FRET signal be- tween Fu and Smo. We then found that FRET between Cos2-YFPC and Smo-CFPC was increased in response to Hh or following the co-transfection of Smo phosphor- mimetic mutants. Moreover, increased FRET was also well correlated with Smo activation (Figure 1C). In ad- dition, we employed CFP and YFP as a control. In both the absence and presence of Hh, CFP and YFP showed a very low FRET signal (Figure 1C). Because Hh can stabilize Smo and increase Smo protein levels, increased FRET between Smo-CFPC and Cos2-YFPC might simply be due to the increase of FRET donor level. To test this possibility, we switched the donor to Cos2-CFPC and the receptor to Smo-YFPC. The results suggested that FRET between Smo and Cos2 increased dramatically with Hh treatment (Figure 1C). A similar change in FRET was found when Smo-CFPC and Cos2-YFPC were expressed Figure 1 Hh induces the recruitment of Cos2/Fu to the membrane through the interaction between Smo and the Cos2/Fu complex. (A) S2 cells were co-transfected with Smo-CFPC, SmoSA-CFPC, SmoSD-CFPC (green) and Cos2-YFPC (red), and Myc-tagged Fu, treated with or without Hh, and immunostained with anti-Myc antibody (blue). In the absence of Hh, only a few Cos2/Fu complexes co-localized with wild-type Smo. In the presence of Hh, the majority of Co2/Fu complexes co-local- ized with wild-type Smo and was recruited to the cell plasma membrane. When SmoSA was co-transfected, Cos2/Fu com- plexes were still localized in the cytoplasm following Hh treatment. When SmoSD was co-transfected, Cos2/Fu complexes were localized on the cell plasma membrane even without Hh treatment. (B) Quantification of Smo co-localized with Cos2 by using Smo mutants to mimic Hh gradient. (C, E) FRET efficiency from the indicated constructs in S2 cells treated with or without Hh-condition medium (mean ± sd, n ≥ 15). (D, F) FRET efficiency between Smo-CFPC/Cos2-YFPC (D), or Cos2-CFPC/ Cos2-YFPC (F), expressed in wing discs (mean ± sd, n ≥ 8). A, A-compartment cells away from the A/P boundary; P, P-com- partment cells; A/P, A-compartment cells adjacent to the A/P boundary. Cell Research | Vol 21 No 10 | October 2011