phosphorylated in response to Hh [13], how this phos- phorylation is regulated remains unknown. We next investigated whether Su(fu) phosphorylation is directly affected by dimerized Fu. We found that Su(fu) is phos- phorylated by dimerized Div-Fu or Fv2-Fu in an in vitro kinase assay (Figure 5C and Supplementary information, Figure S8D). Moreover, dimerized FuK*, which con- tains a Su(fu) binding region, but not FuK, which lacks a Su(fu) binding site, phosphorylated Su(fu), indicating that the binding of Fu with Su(fu) is required for Su(fu) phosphorylation (Figure 5C). To investigate whether phosphorylation of Fu and Cos2 affects the interaction of Cos2/Fu complex with Smo, we examined the co-localization of Smo and Cos2/ Fu. A previous study suggested that Fu activity regu- lated Smo plasma membrane accumulation [15]. We also found that FuG13V could attenuate the Smo cell mem- brane accumulation (Figure 5D). However, we found that Cos2SA (a phosphorylation-defective mutant of Cos2 in which Cos2 amino acid 572 was mutated from Ser to Ala) could not affect the recruitment of the Cos2/Fu complex by Smo after Hh treatment to the plasma mem- brane (Figure 5D). This result suggests that the phos- phorylation status of Cos2 does not affect the interaction between Smo and the Cos2/Fu complex. It also implies that the phosphorylation of Cos2 is likely a downstream event that occurs after the recruitment of Cos2/Fu to the plasma membrane by Smo in the presence of Hh. Dimerized Fu promotes the dissociation of Ci155 from Cos2 and Su(fu) and Ci155 nuclear translocation In the presence of Hh, Ci155 dissociates from the Cos2/Fu complex [22, 30]. To investigate whether Fu dimerization plays a role in this event, we examined the binding between Ci155 and Cos2 in response to a Hh gradient mimicked by Smo phosphor-mimetic mutants, or by dimerized Fu. We found that the binding between Cos2 and Ci155 became weaker with an increased Hh gradient as mimicked by graded Smo activity (Figure 6A). Importantly, dimerized Fu but not FuG13V induced a similar dissociation of Cos2 and Ci155 to the graded Smo activity, suggesting that Hh-induced Fu dimeriza- tion dissociates Ci155 from the Cos2/Fu complex (Figure 6B-6B′). Su(fu), which binds Ci/Gli and inhibits the activity of Ci155/Gli, dissociates from Ci155/Gli in response to a high concentration of Hh [7, 31]. We next investigated the effect of either the Hh gradient or Fu dimerization on the association of Su(fu) and Ci155. We found that low or medium Hh concentrations, which were mimicked by SmoSD1 or SmoSD12, did not affect the association of Su(fu) and Ci155. However, high concentrations of www.cell-research.com | Cell Research Yanyan Zhang et al. npg 1445 Hh, mimicked by SmoSD, enhanced the dissociation of Ci155 from Su(fu) (Figure 6C). Similarly, dimerized Fu caused a dissociation of Ci and Su(fu) (Figure 6D-6D′). In addition, Div-FuK* mutant, but not the Div-FuK mu- tant, promoted the dissociation of Su(fu) and Ci155 (Fig- ure 6D″). Collectively, these data suggest that dimerized Fu phosphorylates Su(fu), leading to the dissociation of Ci155 and Su(fu). Hh not only prevents the processing of Ci155, but also promotes the nuclear translocation of accumulated Ci155 [32-34], and in the presence of high levels of Hh signal- ing, the E3 Roadkill/Hib can inhibit Ci nuclear import [35]. To determine how Hh and Fu dimerization regu- lates Ci155 nuclear translocation, we used LMB to block Ci155 nuclear export and Su(fu) to inhibit Ci155 nuclear translocation and then examined Ci155 accumulation in the nucleus. We found that very little Ci155 entered the nucleus without Hh treatment, but this increased dramati- cally when cells were treated with Hh (Figure 6E). With LMB, almost all Ci155 was located in the nucleus in the absence of Hh, an effect that was blocked by co-trans- fecting Su(fu). Hh treatment or co-transfecting SmoSD antagonized the negative effect of Su(fu) and promoted Ci155 nuclear translocation (Figure 6E and Supplemen- tary information, Figure S9A). The nuclear translocation of Ci155 was gradually enhanced with increased Smo phosphorylation levels and Hh gradient as mimicked by Smo mutants (Figure 6F and Supplementary information, Figure S9A). Similarly, dimerized Fu, but not dimerized FuG13V, promoted Ci155 nuclear translocation (Figure 6G and Supplementary information, Figure S9B). Taken together, these results suggest that Fu dimerization in- duced by Hh promotes the dissociation of Ci155 and Cos2 or Su(fu), and promotes nuclear translocation of Ci155. Dimerization of Fu promotes pathway activation To further determine whether the dimerization of Fu is sufficient to activate the Hh pathway, we examined the activation of the Hh pathway by dimerized Fu in vivo. Because we failed to induce Fu dimerization by feeding the flies AP20187, an approach that has been success- fully employed in a mouse system [24, 25], we used the Div-based Fu dimerization system for in vivo experi- ments. We found that wing imaginal discs expressing UAS-Div-Fu by act>CD2>Gal4, displayed a dpp-lacZ expression in clones at the A-compartment (Figure 7B). In contrast, Div or Div-FuG13V failed to activate dpp- lacZ (Figure 7A and Supplementary information, Figure S10A-S10A″). Moreover, both Div-FuK and Div-FuK* activated dpp-lacZ, with more extensive expression ob- served in those that expressed Div-FuK* (Figure 7C,