Rap1 acts via multiple mechanisms to position Canoe/Afadin and adherens junctions and mediate apical-basal polarity establishment

Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. The mechanisms underlying this remain key issues. Drosophila embryos provide a superb model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (mammalian Afadins homolog) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junctions positioning. Here we define mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane-localization, recruitment to nascent junctions and macromolecular assembly into cables at tricellular junctions. Our data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy/PDZ-GEF is critical for Canoe tricellular junction enrichment but not apical retention. Canoes Rap1-interacting RA-domains mediate adherens junction and tricellular junction recruitment but are dispensable for membrane-localization. Our data also support a role for Canoe-multimerization. These multifactorial inputs all shape Canoe localization, correct Bazooka/Par3 and adherens junction positioning, and thus apical-basal polarity. We integrate existing data into a new polarity establishment model.\n\nAbbreviations used-cat, alpha-catenin; {beta}-cat, beta-catenin; AJ, adherens junction; Arm, Armadillo; Baz, Bazooka; CA, constitutively active; Cno, Canoe; DE-cad, Drosophila E-cadherin; Dzy, Dizzy; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GTP, guanosine triphosphate; IF, immunofluorescence; MIP, maximum intensity projection; RA, Ras-associated; RFP, red fluorescent protein; SAJ, spot adherens junction; shRNA, short hairpin RNA; TCJ, tricellular junction; WT, wildtype