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

Abstract

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.

The Drosophila Afadin and ZO-1 homologs Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling

“…Our recent super-resolution imaging in cultured mammalian MDCK cells suggested Cno's homolog Afadin plays a prominent role at tricellular junctions where three cells meet, reinforcing end-on links between the actomyosin cytoskeleton and cell-cell junctions and thus allowing tricellular junctions to resist mechanical tension generated by actomyosin contractility along the cell border (Choi et al, 2016). These data fit with our earlier work in Drosophila, which revealed Cno is enriched at tricellular junctions and strengthens actomyosinjunction linkages during mesoderm invagination and germband elongation (Sawyer et al, 2009;Sawyer et al, 2011;Bonello et al, 2018). We sought to further explore Cno's role at tricellular junctions in in vivo.…”

Scribble and Discs-large direct adherens junction positioning and supermolecular assembly to establish apical-basal polarity

“…4A,B,brackets), and is especially enriched at TCJs ( Fig. 4G',arrows), where it forms cable-like structures (Bonello et al, 2018). Unlike core AJ proteins, Cno puncta are not found basolateral to SAJs ( Fig.…”

“…However, this is not what we observed. As cellularization proceeds, AJ proteins localize to an apicolateral position in punctate SAJs ( (Bonello et al, 2018), Dlg (Fig. 1G,H) and Scrib (Fig.…”

“…An apical actin-based scaffold thought to anchor Baz and AJs apically and dynein-driven apical transport also play roles (Harris and Peifer, 2005). The actinjunction crosslinker Canoe (Cno=fly Afadin) and its regulator, the GTPase Rap1, act upstream of both Baz and AJ positioning (Bonello et al, 2018;Choi et al, 2013). Activating Rap1 apically positions Cno at nascent SAJs where Cno directs Baz and AJ positioning.…”

Telophase correction refines division orientation in stratified epithelia

“…In Drosophila, Cno is essential for early establishment of apical-basal polarity during cellularization (Bonello, Perez-Vale, Sumigray, & Peifer, 2018;W. Choi, Harris, Sumigray, & Peifer, 2013).…”