Fig. 8. Ptc¹⁴ does not accumulate in endosomes. (A,B) Internalized Red-dextran (5 minutes pulse) at the AP border of a wild-type wing imaginal disc (A) and in a ptc¹⁴ clone (B). (a) Details of the colocalization of Red-dextran positive vesicles and Ptc (green). (b) There is no colocalization of the internalized Red-dextran vesicles with Ptc protein in ptc¹⁴ clone cells. There is reduced Ptc staining in vesicles labeled with Red-dextran (green, arrowheads). Very few Red-dextran vesicles contain appreciable accumulation of Ptc (B, circles) compared with wild-type Ptc (A, circles). (C,D) Extracellular staining of Ptc in a wing disc containing ectopic Ptc^WTGFP (C) and Ptc¹⁴GFP (D) clones using the anti-Ptc antibody, which recognizes one of the extracellular domains of Ptc. Staining is observed in both clones (arrowheads), indicating that a certain amount of Ptc^WTGFP and Ptc¹⁴GFP reaches the plasma membrane. (E,F) Ptc^WTGFP (E) and Ptc¹⁴GFP (F) ectopic clones in the A compartment far from the AP border in wing discs incubated with Red-dextran for 10 minutes. (e) Magnification of E showing the extensive co-localization between Ptc^WTGFP and internalized Red-dextran (circles), indicating that Ptc^WTGFP is endocytosed independently of the presence of Hh. (f) Magnification of F showing very little colocalization between Ptc¹⁴GFP and internalized Red-dextran (arrowheads), indicating that Ptc¹⁴GFP is not efficiently endocytosed. Circles indicate some Red-dextran vesicles with Ptc¹⁴GFP accumulation. The analysis of the Ptc^WTGFP protein content in endocytic vesicles compared with the Ptc¹⁴GFP mutant protein (E,F, columns) shows at least eight times more wild-type Ptc protein in endosomes than Ptc mutant protein (see Materials and methods). Scale bars: 15 µm in A,B; 50 µm in C-F.