Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function

Abstract

Epithelial tissues are defined by polarized epithelial cells that are integrated into tissues and exhibit barrier function in order to regulate what is allowed to pass between cells. Cell-cell junctions must be stable enough to promote barrier function and tissue integrity, yet plastic enough to remodel when necessary. This remarkable ability to dynamically sense and respond to changes in cell shape and tissue tension allows cell-cell junctions to remain functional during events that disrupt epithelial homeostasis including morphogenesis, wound healing, and cell division. In order to achieve this plasticity, both tight junctions and adherens junctions are coupled to the underlying actomyosin cytoskeleton. Here, we discuss the importance of the junctional linkage to actomyosin and how a localized zone of active RhoA along with other Rho GTPases work together to orchestrate junctional actomyosin dynamics. We focus on how scaffold proteins help coordinate Rho GTPases, their upstream regulators, and their downstream effectors for efficient, localized Rho GTPase signaling output. Additionally, we highlight important roles junctional actin-binding proteins play in addition to their traditional roles in organizing actin. Together, Rho GTPases, their regulators, and effectors form compartmentalized signaling modules that regulate actomyosin structure and contractility to achieve proper cell-cell adhesion and tissue barriers.

Keywords: Adherens junction; Arp2/3; F-actin; Formin; Myosin II; Tight junction.

Figure 1. The apical junctional complex (AJC) establishes epithelial barrier function, cell adhesion, and integrity of epithelial tissues

A) A side view schematic of epithelial cells showing the two types of junctions in the AJC, the tight junction (TJ), which establishes the barrier function, and the adherens junction (AJ), which regulates cell-cell adhesion. In the enlarged view below, the architecture of core TJ and AJ proteins is shown (see legend identifying key proteins). Transmembrane proteins facilitate interaction between cells, and scaffolding proteins connect the transmembrane proteins to bundles of contractile F-actin and Myosin II. Note that in this view the bundles of actomyosin are oriented perpendicular to the plane of the cross-section. B) A top view schematic of epithelial cells. In the enlarged views below, one model for the organization of actomyosin is depicted with bundled antiparallel F-actin, crosslinked by α-actinin, and decorated with Myosin II motors.

Figure 2. Rho GTPase cycle, key effectors, and resulting actin organization

A) Typical Rho family GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. GEFs activate GTPases by promoting the exchange of GDP for GTP, while GAPs inactivate GTPases by stimulating GTP hydrolysis. Rho GDI sequesters Rho-GDP in the cytoplasm, protecting it from degradation and preventing its activation. In the active conformation, Rho GTPases activate effector proteins leading to the biological output, which depending on the Rho GTPase involved, results in specific, localized effects on the cytoskeleton. B) RhoA-GTP signals through its effectors, formins and ROCK, to promote the formation of actomyosin contractile arrays. C) Rac1-GTP and Cdc42-GTP signal through their effectors – WAVE and N-WASP, respectively – to promote Arp2/3-mediated branched actin structures. In some cases, Rac1 and Cdc42 can also trigger formin activity to promote unbranched actin polymerization.

Figure 3. Possible models and key players in junction formation, maturation, and stability

A) Cells migrate towards each other in a Rac1- and Cdc42-dependent manner. Activity of these GTPases establishes an Arp2/3 branched F-actin network that pushes cell membranes together to establish cell-cell contacts. Cdc42-GTP also generates protrusive filopodia that are important for cell-cell contact formation (not shown). B) Junction elongation and early junction tension are promoted by Rac1 and Cdc42. The branched F-actin network is converted into a linear contractile actomyosin array through RhoA activation and by Coronin1B displacing Arp2/3 complexes, creating more flexible hinge points to linearize F-actin. C) Stable junctions are maintained through proper RhoA activity, which promotes contractile F-actin through stimulating F-actin polymerization via formins and Myosin II activity via ROCK. A population of Arp2/3-dependent F-actin that is rapidly turning over also contributes to stable junctions (not shown).

Figure 4. Scaffolding proteins dictate Rho GTPase signaling outcomes

A-C) Speculative models of how three scaffold proteins may facilitate proper Rho GTPase signaling output. In each case, the scaffold protein is shown in turquoise on the left, and key binding partners of the scaffold protein are listed. A) A speculative model of how Cingulin might regulate Rho signaling output. The head domain of Cingulin can bind F-actin and Myosin II, anchoring it to the cytoskeleton. Under certain conditions (e.g. as a result of phosphorylation or ZO-1 binding, shown here), Cingulin may reorient, giving GEFs (e.g. p114RhoGEF, shown here) and GAPs that are bound to Cingulin’s rod domain access to their target GTPases. B) p120 catenin is a hub for RhoA/Rac1 signaling. When bound to E-cadherin, p120 catenin prevents E-cadherin endocytosis, contributing to junction maturation and stability, and promotes RhoA inactivation by recruiting p190RhoGAP-B. In its cytoplasmic form, p120 catenin promotes Rac1 activation (and Cdc42 activation, not shown here) by recruiting the GEF Vav2. p120 also performs GDI-like RhoA inactivation by binding to RhoA-GDP, preventing its activation. C) Junctional Anillin may act to coordinate RhoA signaling in a tension-sensitive manner, as it does during cytokinesis. Under low or moderate tension, Anillin acts to scaffold active RhoA with formins and junctional actomyosin. We speculate that a tension-sensitive conformation of Anillin, may recruit p190RhoGAP-A to promote RhoA inactivation in order to fine-tune RhoA signaling and maintain balanced tension at the AJC.