Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell–cell junctions to allow dynamic remodeling while resisting forces generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional–cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction–cytoskeletal linkage during gastrulation. Canoe’s mammalian homologue Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We take these insights back to the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found that Canoe helps cells maintain junctional–cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation, and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe, many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homologue Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest that Canoe and Polychaetoid stabilize Bazooka/Par3 at cell–cell junctions, helping maintain balanced apical contractility and tissue integrity.

Mutations in the KRAS oncogene are found in more than 90% of patients with pancreatic ductal adenocarcinoma (PDAC), with Gly-to-Asp mutations (KRASG12D) being most common. Here, we tested the efficacy of a small molecule KRASG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14d. Concomitant with tumor cell apoptosis and proliferative arrest, drug treatment led to marked shifts in the tumor microenvironment (TME), including changes in fibroblasts, matrix, and macrophages. T cells were necessary for MRTX1133’s full anti-tumor effect, and T cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KRASG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies.

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. Defining the mechanisms underlying polarity establishment remains a key issue. embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction 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 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 crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe 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 shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. We integrate the existing data into a new polarity establishment model.

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 Afadin’s 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. Canoe’s 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.Abbreviations usedα-cat, alpha-catenin; β-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

During embryonic development, a simple ball of cells re-shapes itself into the elaborate body plan of an animal. This requires dramatic cell shape changes and cell movements, powered by the contractile force generated by actin and myosin linked to the plasma membrane at cell-cell and cell-matrix junctions. Here, we review three morphogenetic events common to most animals: apical constriction, convergent extension and collective cell migration. Using the fruit fly Drosophila as an example, we discuss recent work that has revealed exciting new insights into the molecular mechanisms that allow cells to change shape and move without tearing tissues apart. We also point out parallel events at work in other animals, which suggest that the mechanisms underlying these morphogenetic processes are conserved.

Embryonic morphogenesis is powered by dramatic changes in cell shape and arrangement, driven by the cytoskeleton and its connections to adherens junctions. This requires robust linkage, allowing morphogenesis without disrupting tissue integrity. The small GTPase Rap1 is a key regulator of cell adhesion, controlling both cadherin-mediated and integrin-mediated processes. We have defined multiple roles in morphogenesis for one Rap1 effector, Canoe/Afadin, which ensures robust junction-cytoskeletal linkage. We now ask what mechanisms regulate Canoe and other junction-cytoskeletal linkers during  Drosophila morphogenesis, defining roles for Rap1 and one of its guanine nucleotide exchange factor (GEF) regulators, Dizzy. Rap1 uses Canoe as one effector, regulating junctional planar polarity. However, Rap1 has additional roles in junctional protein localization and balanced apical constriction—in its absence, Bazooka/Par3 localization is fragmented, and cells next to mitotic cells apically constrict and invaginate, disrupting epidermal integrity. In contrast, the GEF Dizzy has phenotypes similar to but slightly less severe than Canoe loss, suggesting this GEF regulates Rap1 action via Canoe. Taken together, these data reveal that Rap1 is a crucial regulator of morphogenesis, likely acting in parallel via Canoe and other effectors, and that different Rap1 GEFs regulate distinct functions of Rap1.

During morphogenesis cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting force generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation.Canoe's mammalian homolog Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We took these insights back into the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found Canoe helps cells maintain junctional-cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation and neuroblast invagination, or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homolog Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest Canoe and Polychaetoid stabilize Bazooka/Par3 at cell-cell junctions, helping maintain balanced apical contractility and tissue integrity.