Fig. 3.

Fluorescent knock-in alleles of DE-Cadherin and crumbs. (A) Protein domain structures of DE-Cad, Crb, and their fluorescent knock-in alleles. In all DE-Cad knock-in alleles, the fluorescent proteins were fused to the C terminus. In 3 Crb::GFP alleles, the GFP was inserted at 2,121 aa (Crb:GFP-A), 2,156 aa (Crb::GFP-B), and 2,189 aa (Crb::GFP-C), respectively. Note that not all of the 30 EGF repeats of Crb are drawn. (B–E) Subcellular localization patterns of DE-Cad::GFP, DE-Cad::PAGFP (photoactivatable GFP), DE-Cad::mTomato, DE-Cad::mCherry in live pupal (B, D, and E) or late embryonic (C) epithelia. All alleles rescued DE-Cad founder lines and were homozygous-viable, but only DE-Cad::GFP and DE-Cad::PAGFP showed clean localization at the adherens junction (B and C). Note the intracellular aggregates of DE-Cad::mTomato and DE-Cad::mCherry in (D) and (E). In (C) the yellow boxes highlight the region before (Top) and after (Bottom) the UV laser irradiation in the same sample. DE-Cad::PAGFP is only fluorescent after UV irradiation. DE-Cad::GFP knock-in homozygotes provide a clean and homogenous background of DE-Cad::GFP, whose expression level is virtually identical to the DE-Cad in wild type (see Fig. 2). (F–H) Subcellular localization of Crb::GFP-A, Crb::GFP-B, and Crb::GFP-C in live embryonic epithelia (stage 11). crb::GFP-A and crb::GFP-C complemented crb^GX24 and were homozygous viable. They show normal localization along the apical-lateral boundary of the epithelial cells (F and H). In contrast, Crb::GFP-B shows a disrupted localization pattern (G). crb::GFP-B failed to complement crb^GX24 and crb^11A22, and is homozygous lethal. All images were taken as the tangential view of the epithelia.