Smo C-terminal region from amino acids 656 to 755 self- interacts with the region from amino acids 860 to 1 035 [17]. Therefore, in the absence of Hh, the binding region of Cos2 on the Smo C-tail is likely masked by the self- interaction between these two Smo C-tail regions. As the interaction between Fu and the C-terminal region of Smo in the absence of Hh may place Cos2 in close proxim- ity to the third cytoplasm loop of Smo, we further tested this possibility by inserting a CFP into the third internal loop of SmoWT, SmoSA or SmoSD to generate SmoL3- CFP, SmoL3SA-CFP and SmoL3SD-CFP, respectively, and co-transfecting them with Cos2-YFPC in S2 cells. We found that FRET between SmoL3-CFP and Cos2-YFPC changed from high to low along with Hh stimulation. FRET between SmoL3SA-CFP and Cos2-YFPC was high and FRET between SmoL3SD-CFP and Cos2-YFPC was low (Figure 2E). These results indicate that Cos2 is close to the third internal loop of Smo when Fu interacts with the C-terminal end of Smo in the absence of Hh. Hh promotes Fu dimerization As both Smo and Cos2 form dimers, and the Cos2/Fu complex interacts with Smo following Hh treatment, we investigated whether Fu forms a dimer in response to Hh. We examined FRET efficiency by co-transfecting Fu-CF- PN and Fu-YFPN in S2 cells together with Smo and Cos2 with or without Hh treatment. FRET between Fu-CFPN and Fu-YFPN was low in the absence of Hh but increased dramatically in the presence of Hh. The different phos- phor-mimetic forms of Smo progressively increased the basal FRET between Fu-CFPN and Fu-YFPN in a graded manner (Figure 3A). In wing imaginal discs, Hh also in- duced a significant increase in FRET between Fu-CFPN and Fu-YFPN at the P-compartment (we used Ms1096 to drive Fu protein expression in the wing pouch) and the A/P boundary (to generate a more convincing result, we used Ptc gal4 to drive Fu protein expression at the A/P boundary in a separate experiment), compared with that at the A-compartment (Figure 3B and Supplementary in- formation, Figure S5). We also measured FRET between Fu-CFPC and Fu-YFPC in S2 cells with similar results (Supplementary information, Figure S6). Therefore, Fu forms a dimer in response to Hh or graded Smo activity. We next examined whether Fu dimerization is dependent on Smo and/or Cos2. We found that FRET efficiency be- tween Fu-CFPN and Fu-YFPN was decreased when Cos2 or Smo expression was inhibited by RNAi, suggesting that the dimerization of Fu induced by Hh or graded Smo activity is Smo and Cos2 dependent (Figure 3C-3D and Supplementary information, Figure S7A-S7B). Cos2 is a kinesin-related protein whose motor domain is important for its transportation along the microtubules www.cell-research.com | Cell Research Yanyan Zhang et al. npg 1441 [20-23]. We generated a Cos2-YFPC mutant, Cos2ΔMD- YFPC, in which the motor domain was deleted, and examined the co-localization of Smo and Cos2ΔMD. We found that Cos2ΔMD still co-localized with Fu but this Cos2ΔMD/Fu complex failed to be recruited to the membrane following Hh treatment (Figure 3E and 3E′). Moreover, in the presence of Hh, FRET between Smo- CFPC and Cos2ΔMD-YFPC was significantly lower than that between Smo-CFPC and Cos2WT-YFPC (Figure 3F). Therefore, Cos2 transportation mediated by its motor domain is important for the recruitment of the Cos2/Fu complex to the plasma membrane by Smo in response to Hh signaling. Given the fact that Cos2 moves along mi- crotubules and is released in the presence of Hh [20, 21, 23], our results imply that this movement of Cos2 along microtubules may bring Fu in proximity to Smo and then dissociate from microtubules and interact with Smo in the presence of Hh. We next investigated whether Fu plays a role in the recruitment of the Cos2/Fu complex to the membrane by Hh. We observed a 33% reduction of FRET between Smo and Cos2 when endogenous Fu expression is inhib- ited by RNAi (Figure 3G and Supplementary informa- tion, Figure S7C). Given that the loss of endogenous Fu did not affect the co-localization of Cos2 and Smo on the plasma membrane following Hh treatment (Figure 2A), Fu may only play a minor role in the recruitment of the Cos2/Fu complex by Smo. Dimerized Fu activates the Hh pathway independently of Smo and Cos2 As the formation of Fu dimer is regulated by Hh and requires Smo and Cos2, Smo may transduce the Hh by inducing Fu dimerization, in turn activating the Hh path- way. To test this hypothesis, we investigated whether dimerized Fu could activate downstream gene expres- sion by generating two Fu dimerization systems. The FKBP-based Fu dimerization system is dependent on a synthetic dimerizing agent (AP20187) that crosslinks two molecules carrying FKBP domains based on its sub- nanomolar binding affinity [24, 25]. Fv2-Fu contains two FK506-binding domains and can be induced to form a dimer or oligomer upon AP20187 treatment. As the FKBP-AP20187 system is not suitable for in vivo experi- ments, we employed DivIVA protein to generate another Fu dimerization system. DivIVA (abbreviated to Div) is a coil-coiled domain-containing protein that forms consti- tutive dimers or oligomers [26]. We therefore generated a Div-Fu fusion protein to examine the in vivo function of dimerized Fu. In addition, several Fu mutants were also generated and examined using these two dimerization systems. FuG13V is a kinase-dead and non-functional