Rap1 regulates apical contractility to allow embryonic morphogenesis without tissue disruption and acts in part via Canoe-independent mechanisms

Abstract

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.

Rap1 promotes epithelial integrity and cell viability in a growing tissue

“…While Rap1 has known roles in the morphogenesis of diverse epithelia (Kim et al, 2022; Knox and Brown, 2002; Wang et al, 2013), few studies have analyzed how Rap1 maintains epithelia during tissue growth. To address this, we inhibited Rap1 activity in the follicle cells using a validated dominant negative Rap1 construct, UAS-DN-Rap1 N17 (DN-Rap1 N17 ), whose expression causes phenotypes that strongly resemble loss of Rap1 or Rap1 RNAi knockdown phenotypes in the embryo and ovary (Boettner et al, 2003; Perez-Vale et al, 2022; Sawant et al, 2018). DN-Rap1 N17 was driven by an epithelial-specific GAL4 driver, c306 -GAL4 (Fig.…”

The Dilute domain of Canoe is not essential for Canoe’s role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis

“…In this way, it resembled two of the mutants we examined earlier, cno Δ PDZ and cno Δ FAB (Perez-Vale et al, 2021). While the tandem RA domain cassette and its Rap1 binding partner are important for initial Cno localization to AJs as they first assemble, they become dispensable for AJ localization after gastrulation onset (Perez-Vale et al, 2023; Perez-Vale et al, 2021). Thus, four of the five folded domains present in Cno, along with its conserved C-terminal FAB, are each singly non-essential for AJ recruitment, despite their conservation from Drosophila to mammals, over ∼600 million years of evolutionary time.…”

“…At their amino terminus are two Ras-association (RA) domains, which bind to the small GTPase Rap1 (Boettner et al, 2000). Rap1 “activates” Cno by mechanisms we are only starting to understand (Boettner et al, 2003; Bonello et al, 2018; Perez-Vale et al, 2023). The central PDZ domain binds several partners, including the transmembrane junctional proteins E-cadherin (Ecad) and Nectins/Echinoid (Sawyer et al, 2009; Takahashi et al, 1999; Wei et al, 2005).…”

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins

“…However, in M/Z pyd mutants Cno remained somewhat enriched on AP borders (Fig. 2E vs F; quantified in 2I), contrasting with the reversal of Cno planar polarity seen after loss of its regulator Rap1 (Perez-Vale et al, 2023) or deletion of Cno's RA domain (Perez-Vale et al, 2021). Thus, Pyd restrains planar polarity of some but not all AJ proteins.…”