Fig. 2. Frizzled2 stimulates Wingless internalization. (A-A''',B-B''') Colocalization of Wingless and Frizzled2 or Frizzled2GPI in an endocytic compartment. (A) Imaginal discs were isolated from dpp-Gal4 UAS-Frizzled2-FLAG and bathed in 3 kDa dextran for 10 minutes. After a 20-minute chase, they were fixed and stained. (A) Low magnification of imaginal disc. Boxed area shown at high magnification in A',A'''. Anti-Wingless is shown in A and A', anti-FLAG (Frizzled2) in A'' and Texas-red Dextran in A'''. (B) Imaginal disc expressing Frizzled2GPI under the control of dpp-Gal4 and labelled with anti-Wingless (B,B'), anti-Myc (B'') and dextran as described (B'''). Wingless is strongly stabilized (compare the extent of stabilization, indicated by double-headed arrows in A,B). (B'-B''') High magnification of the boxed area in B. There is reduced colocalization of Dextran, Frizzled2GPI and Wingless. (C) Binding of GFP-Wingless to S2R+ cells transfected with Frizzled2-FLAG. The transfected cells in this field of view are indicated by white arrows. Binding was performed at 4°C, hence the round appearance of the cells. GFP-Wingless in this and all subsequent experiments is detected with an anti-GFP antibody. (D,D') Upon shifting to 25°C, GFP-Wingless (D') is internalized in Frizzled2-FLAG-positive structures (D, detected with anti-Flag). Wingless internalization is also seen in untransfected cells (asterisk), although at a much reduced level (total fluorescence of the untransfected cell is 36% that of the transfected cell). In D' (and F'), residual extracellular staining was stripped so that only internalized epitopes are revealed. (E) Binding at 4°C of GFP-Wingless to cells transfected with Frizzled2GPI. (F,F') In cells transfected with Frizzled2GPI (F, detected with anti-Myc), little internalization of GFP-Wingless (F') is seen upon shifting to 25°C (the difference in mean fluorescence between transfected and untransfected cells is over fivefold higher in the Frizzled2 cells shown than in the corresponding Frizzled2GPI cells). Weakly expressing cells do internalize GFP-wingless (not shown) perhaps because this fusion protein can cooperate with endogenous proteins that contain an internalization signal. Scale bars: 10 µm.