The dual Ras Association (RA) Domains of Drosophila Canoe have differential roles in linking cell junctions to the cytoskeleton during morphogenesis

During embryonic development and adult homeostasis epithelial cells must change shape and move without disrupting the tissue’s dynamic architecture. This requires robust linkage of cell-cell adherens junctions to the force-generating actomyosin cytoskeleton. Drosophila Canoe and mammalian Afadin play key roles in this linkage. One central task for the field is defining how upstream inputs from Ras-family GTPases regulate Canoe and Afadin. They are unusual in that they share two tandem Ras-association (RA) domains, which, when deleted, virtually eliminate Canoe function. Previous work in vitro suggested RA1 and RA2 differ in their ability to bind GTPases, but their individual functions in vivo remain unknown. Combining bioinformatic and biochemical approaches, we find that both RA1 and RA2 bind to active Rap1 with similar affinities, and that conserved N-terminal extensions play a role in binding. We created Drosophila canoe mutants to test RA1 and RA2 function in vivo. Despite their similar affinities for Rap1, RA1 and RA2 play strikingly different roles. Deleting RA1 virtually eliminates Canoe function in morphogenesis, while mutants lacking RA2 are viable and fertile but have defects in junctional reinforcement in embryos and during pupal eye development. These data significantly expand our understanding of how adherens junction:cytoskeletal linkage is regulated.