30 min at room temperature. The fixed wings or discs were incubated with blocking solution (PBS containing 0.2% (w/v) BSA and 0.1% (v/v) Triton X-100) at 4uC for 1 h and then incubated with the following primary antibodies: rat anti-DE-cadherin (1:50 DCAD2 DSHB), mouse anti-Flamingo (1:20; a gift from T. Uemura), mouse anti-Armadillo (1:200; N2 7A1 DSHB), mouse anti-Discs large (1:200; number 4F3 DSHB), mouse anti-FasIII (1:200; 7G10 DSHB), mouse anti-b-integrin (1:50;CF.6G11 DSHB), rabbit anti-GFP (1:300; MBL), rat anti-GFP (1:300; MBL), rat anti-Rbn5 (1:300 a gift from T. Tanaka), rabbit anti- Rbn5 (1:300; a gift from T. Tanaka), mouse anti-120K (1:200; Calbiochem), rabbit anti-Rab5 (1:50, a gift from M. Gonzalez- Gaitan), rabbit anti-Rab7 (1:300; a gift from T. Tanaka), rabbit anti-Rab11 (1:1000; a gift from S.M. Cohen), rabbit anti-Nuf (1:3000; a gift from W. Sullivan) and rabbit anti-GM130 (1:200; Sigma) for 4uC overnight. After the samples were washed with blocking solution five times, the following appropriate secondary antibodies were added and left for 2 h at 4uC; Cy3 conjugated anti-mouse IgG (1:200; Amersham Pharmacia), Cy3 conjugated anti-rat IgG (1:200; Amersham Pharmacia), Cy3 conjugated anti- rabbit IgG (1:200; Amersham Pharmacia), Alexa Fluor 488- conjugated anti-mouse IgG (1:200; Molecular Probes), Alexa Fluor 488-conjugated anti-rat IgG (1:200; Molecular Probes), Alexa Fluor 488-conjugated anti-rabbit IgG (1:200; Molecular Probes) TO-PRO3 (1:300; Molecular Probes) and Rhodamine phalloidin (1:300; Molecular Probes). All images were obtained using an Olympus FV-500 confocal microscope and processed using Adobe Photoshop. TR-D incorporation assay Larva wing discs (3rd) were dissected and then incubated with 0.5 mM TR-D (MW 3000, Molecular Probes) in Schneider’s medium (GIBCO-BRL) containing 10% (v/v) FCS, 50 U/ml penicillin, and 50 mg/ml streptavidin for 5 min at 4uC [Pulse]. After washing twice with ice-cold Schneider’s medium, the wing discs were then incubated for 5, 20, 40 and 60 min. with fresh medium at 26.5uC, respectively [Chase]. The wing discs were fixed with 4% (w/v) PFA-PBS solution for 30 min at room temperature and observed using an Olympus FV-500 confocal microscope and processed with Adobe Photoshop. Lysotracker staining Larvae wing discs (3rd) were dissected and then stained for 30 sec with Lysotracker red DND-99 (Molecular Probes) diluted 1:1000 in PBS. Mounting was performed in 80% glycerol in PBS, and the samples were directly visualized by confocal microscopy. References 1. Apodaca G, Katz LA, Mostov KE (1994) Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J Cell Biol 125: 67–86. 2. Parton RG, Prydz K, Bomsel M, Simons K, Griffiths G (1989) Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes. J Cell Biol 109: 3259–3272. 3. Brown PS, Wang E, Aroeti B, Chapin SJ, Mostov KE, et al. (2000) Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling. Traffic 1: 124–140. 4. Bomsel M, Prydz K, Parton RG, Gruenberg J, Simons K (1989) Endocytosis in filter-grown Madin-Darby canine kidney cells. J Cell Biol 109: 3243–3258. 5. Herman PK, Emr SD (1990) Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol 10: 6742–6754. 6. Hiles ID, Otsu M, Volinia S, Fry MJ, Gout I, et al. (1992) Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell 70: 419–429. 7. Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, et al. (1993) Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260: 88–91. PI3K & Rbsn-5 in Drosophila Electron microscopy Thirty hour APF staged pupae wings were dissected and fixed with 2.5% (v/v) Glutaldehyde (GA), 2% (w/v) sucrose, 1% (w/v) tannic acid in PBS for 30 min at room temperature and then at 4uC overnight. The samples were then re-dissected to remove the wing cuticles and subjected to post-fixation with 1.0% (v/v) osmium oxide for 1 h on ice. The wings were then dehydrated and embedded in epoxy resin (Epon812, TAAB). Ultrathin sections were obtained using a ultramicrotome (Leica Ultracut UCT), and staining with uranyl acetate and lead citrate. Specimens were observed using a transmission electron microscope (JEOL JEM- 1230). For pre-embedding immunoelectron microscopy, dissected pupae were fixed in a mixture of 4.0% (w/v) PFA and 0.01% (v/v) GA in PBS for 30 min at room temperature and then for 1 h at 4uC. The samples were re-dissected with 150 mM (w/v) glycine in PBS to remove the wing cuticles, and incubated with blocking solution (PBS containing 0.1% (w/v) glycine, 5% (v/v) normal goat serum, 0.1% (w/v) saponin, 0.05% (v/v) Triton X-100 and 0.1% (v/v) gelatin) for 30 min at 4uC. Specimens were incubated with a primary antibody solution (PBS containing 1% (v/v) normal goat serum, 0.1% (w/v) saponin, 0.05% (v/v) Triton X-100, 0.1% (v/v) gelatin and rat anti-GFP (1:10; MBL)) overnight at 4uC and then incubated with anti-rat antibodies conjugated to 1.4 nm gold particles (Nanoprobes) for 3 h at room temperature. The wings were re-fixed with 2% GA in PBS for 15 min at room temperature. They were then treated using an HQsilver kit (Nanoprobes) for 7–10 min at room temperature in the dark. After post-fixing with 0.5% OsO4 for 45 min on ice, the wings were dehydrated and embedded into Epon812 resin. Acknowledgments We thank S.M. Cohen, S. Eaton, M. Gonzalez-Gaian, K, Matuno, W. Sullivan, the Bloomington Stock Center, the Genetic Strains Research Center of National Institute of Genetics, the Drosophila Genetic Resource Center (Kyoto) and the Developmental Studies Hybridoma Bank for fly strains of antibodies, and T. Saitoh, and Y. Hinohara, for their technical supports. We also gratefully acknowledge Y. Shimada, T, Uemura and members of the S.G. Laboratory for their helpful discussions and critical comments. Author Contributions Conceived and designed the experiments: SG. Performed the experiments: MA YS WA SG. Analyzed the data: MA SG. Contributed reagents/ materials/analysis tools: TT KT RU AN. Wrote the paper: MA TT AN SG. 8. Stack JH, Herman PK, Schu PV, Emr SD (1993) A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. Embo J 12: 2195–2204. 9. Gillooly DJ, Morrow IC, Lindsay M, Gould R, Bryant NJ, et al. (2000) Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. Embo J 19: 4577–4588. 10. Christoforidis S, Miaczynska M, Ashman K, Wilm M, Zhao L, et al. (1999) Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat Cell Biol 1: 249–252. 11. Jones AT, Clague MJ (1995) Phosphatidylinositol 3-kinase activity is required for early endosome fusion. Biochem J 311: 31–34. 12. Li G, D’Souza-Schorey C, Barbieri MA, Roberts RL, Klippel A, et al. (1995) Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci U S A 92: 10207–10211. 13. Spiro DJ, Boll W, Kirchhausen T, Wessling-Resnick M (1996) Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro. Mol Biol Cell 7: 355–367. 14. Burda P, Padilla SM, Sarkar S, Emr SD (2002) Retromer function in endosome- to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J Cell Sci 115: 3889–3900. PLoS ONE | www.plosone.org 11 October 2009 | Volume 4 | Issue 10 | e7306