Crb promotes DaPKC phosphorylation on Lgl in vivo. (A and B) Drosophila lgl::GFP or lgl^S6A::GFP follicular epithelial cells overexpressing Crb were immunostained for GFP (green), DaPKC (red), and Crb (magenta). Images in A are in tangential view and were sectioned below the apical surface of follicular cells where Crb and aPKC normally are absent. Images in B are in cross section view of follicular epithelial cells, showing overexpressed Crb expanded into the lateral PM along with DaPKC. Cells overexpressing Crb are highlighted by asterisks in green-channel images. (C) Wild-type lgl::GFP embryos and embryos of lgl::GFP UAS-Crb/Mat-Gal4 or lgl^S5A::GFP UAS-Crb/Mat-Gal4 were immunostained for GFP (green), Crb (red), and aPKC (magenta). All embryonic epithelial cells were in cross-section view. Note the loss of Lgl::GFP, but not Lgl^S5A::GFP, from the PM under Crb overexpression driven by Mat-Gal4 (see Materials and methods). (D) Wild-type lgl::GFP embryos and lgl::GFP; crb^−/− mutant embryos were immunostained for GFP (green), Crb (red), and aPKC (magenta). In crb^−/− embryos, red channel was overexposed to confirm no detectable expression of Crb. In crb^−/− embryonic epithelial cells, both Lgl and DaPKC became localized all around PM. (E and F) Lgl::GFP remained on PM in Crb-overexpressing cells that also expressed DaPKC-RNAi (E) or par-6-RNAi (F). (G) Lgl::GFP was severely lost from PM in follicular cells expressing DaPKCΔN. In E–G, cells expressing DaPKC-RNAi, par-6-RNAi or DaPKCΔN are marked by RFP expression (see Materials and methods). Scale bars: 5 µm.