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During development cells must change shape and move without disrupting dynamic tissue 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 the regulation of such linkages. One central task for the field is defining mechanisms by which upstream inputs from Ras-family GTPases regulate Canoe and afadin. These proteins are unusual in sharing two tandem Ras-association (RA) domains - RA1 and RA2 - which when deleted virtually eliminate Canoe function. Work in vitro has suggested that RA1 and RA2 differ in GTPase affinity, 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 their conserved N-terminal extensions enhance 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, whereas 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 the regulation of adherens junction-cytoskeletal linkages. [ABSTRACT FROM AUTHOR]

This article is an interview with the first authors of a paper published in the Journal of Cell Science. The authors, Emily D. McParland and T. Amber Butcher, conducted research on the Drosophila Canoe protein and its role in linking cell junctions to the cytoskeleton during embryonic development. They found that a specific domain of the Canoe protein, called the Dilute domain, is not essential for this linkage but supports the robustness of morphogenesis. The authors also discuss the challenges they faced during the project and their motivations for pursuing a career in science. [Extracted from the article]

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including its C-terminal conserved motifs, are important for protein function. These data provide the foundation for further analysis of IDR function.