in fly wing imaginal discs (Figure 1D and Supplemen- tary information, Figure S1). Collectively, our data indi- cate that, in response to a Hh gradient and Smo activity, the Cos2/Fu complex is recruited to the cell membrane through an interaction between Smo and Cos2. Cos2 is a kinesin-related protein that may form a dim- er [20, 21]. We measured FRET changes between Cos2- CFPC (Cos2 tagged CFP at its C terminus) and Cos2-YF- PC (Cos2 tagged YFP at its C terminus) to investigate the dimerization of Cos2. We found that Cos2 formed dimers in both S2 cells and wing imaginal discs that were not dependent on the presence of Hh. However, Cos2-Cos2 FRET was decreased with Hh treatment, suggesting that in the presence of Hh, the recruitment of the Cos2/Fu complex to Smo may induce a conformational change of the Cos2 dimer (Figure 1E-1F and Supplementary infor- mation, Figure S2). The Cos2/Fu complex switches the binding region on Smo in the presence of Hh signal To further investigate which region of the Smo C-tail contributes to the interaction between Smo and the Cos2/ Fu complex, we examined the co-localization of Smo with Cos2 or Fu under different conditions. First, we co- expressed Smo-CFPC and Cos2-YFPC when endogenous Fu was knocked down (Figure 2A, 2A’ and Supplemen- tary information, Figure S3A), or we co-transfected with Smo-CFPC and Fu-YFPN in the absence of endogenous Cos2 (Figure 2B, 2B’, Supplementary information, Figure S3B). We found that, in the presence of Hh, the overexpression of Fu or the knockdown of endogenous Fu did not affect the co-localization of Smo with Cos2 on the membrane. However, in the absence of Hh, the percentage of Smo that co-localized with Cos2 decreased in the endogenous Fu knockdown sample compared with the overexpressed Fu sample (Figure 2A and 2A’). In contrast, in the absence of Hh, the co-localization of Fu and Smo in the endogenous Cos2 knockdown condition was greater than the co-localization of Cos2 and Smo in the endogenous Fu knockdown condition (Figure 2A, 2A’, 2B and 2B’). In the presence of Hh, the Smo plasma membrane localization was decreased in the absence of Cos2 (Figure 2B, compare Smo plasma membrane lo- calization with or without Cos2 RNAi in the presence of Hh), and the majority of Fu protein failed to co-localize with Smo on the plasma membrane in the absence of Cos2 (Figure 2B and 2B’). These results, together with the results shown in Figure 1A, imply that Fu contributes to the partial co-localization of the Cos2/Fu complex with Smo in the absence of Hh, whereas Cos2 is respon- sible for the recruitment of the Cos2/Fu complex to the plasma membrane by activated Smo in the presence of www.cell-research.com | Cell Research Yanyan Zhang et al. npg 1439 Hh. Although previous research had provided some bio- chemical evidence for the binding region of Smo with Cos2 [11], the physiological evidence for the interacting region of Smo with Cos2 is lacking. As the binding of the Cos2/Fu complex with Smo may be due to a positional switch in response to Hh, we attempted to determine the region of the Smo C-tail that contributes to its binding with the Cos2/Fu complex. In Smo C-tail, a region of three phosphorylation clusters (amino acids 661 to 818), is important for Smo activity, while the last 59 amino acids of Smo are critical for its binding to Fu [9, 19]. We generated two Smo mutants, SmoΔ974 and SmoΔ818, in which the Smo C-tail fragment from either amino acid 974 or 818 was deleted. We found that, with or without Hh, Fu failed to co-localize in these two Smo mutants when the endogenous Cos2 was knocked down. In ad- dition, no co-localization between Fu and SmoSDΔ974 or SmoSDΔ818 was observed under the same condi- tion (Figure 2C). With co-transfected Cos2, however, Fu co-localized with SmoSDΔ974 or with SmoΔ974 when Hh was present (Supplementary information, Fig- ure S4A). Fu failed to co-localize with SmoSDΔ818 or with SmoΔ818 regardless of the presence or absence Hh (Supplementary information, Figure S4A). These data suggest that Cos2 likely binds to a different region of the Smo C-tail, which is important for the binding of the Cos2/Fu complex to Smo in response to Hh signaling, and its binding region is different from Fu. We further dissected the specific region of Smo that is responsible for Cos2 binding. In the absence of Hh, Cos2 did not co- localize with wild-type Smo or Smo mutants when the endogenous Fu was knocked down (Figure 2A, 2D and Supplementary information, Figure S4B). In the presence of Hh, however, Cos2 co-localized with both SmoΔ974 and SmoSDΔ974 on the cell membrane regardless of the level of Fu. In contrast, Cos2 failed to co-localize with SmoSDΔ818 or with SmoΔ818 in the presence of Hh, suggesting that amino acids 818-974 of Smo are impor- tant for the physiological interaction between Smo and Cos2 in the presence of Hh (Figure 2D and Supplemen- tary information, Figure S4B). The FRET result in Figure 1C also suggests that the C-terminal region of Smo is far from the Cos2 C-terminus in the absence of Hh. Taken together, these data suggest that the Cos2/Fu complex interacts with Smo at different regions in the presence or absence of Hh. In the presence of Hh, Cos2 mediates the interaction between the Cos2/Fu complex and Smo by binding to the region of Smo between amino acids 818 and 974; in the absence of Hh, the C-terminal part of Smo (amino acids 974-1 035) interacts with Fu. The C-tail of Smo keeps a closed and inactivated conformation in the absence of Hh. In this condition, the