Immediate-Early Signaling Induced by E-cadherin Engagement and Adhesion

Abstract

Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusionmediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.Members of the cadherin family of Ca 2ϩ -dependent cell adhesion proteins initiate cell adhesion by trans pairing of cadherins on opposing cell surfaces (1, 2) and then stabilize adhesion by accumulating in the plane of the membrane (3-6) by a process thought to involve the actin cytoskeleton. Live cell analysis of asynchronous cell-cell contacts indicates that initial interactions occur opportunistically between opposing lamellipodia formed as cells crawl. Subsequent expansion of the contact occurs through interactions between additional lamellipodia confined to the perimeter of the contact, whereas lamellipodia activity generally decreases within the contact (6 -8). However, many questions remain. It is unclear whether E-cadherin engagement results in or is the consequence of downstream activation of membrane and actin dynamics, whether E-cadherin accumulation is an active process driven by actin assembly, or how lamellipodia activity is locally regulated during cell-cell adhesion.Actin and membrane dynamics mediated by Rho family GTPases have been implicated in regulating lamellipodia activity and cadherin adhesion (5, 9 -12). Reports show that Rac1 is activated upon E-cadherin adhesion (9, 13, 14) and co-localized with E-cadherin after 30 min (11), and that products of PI 4 3-kinase (8, 15-17), PI(3,4)-bisphosphate and PI(3,4,5)-triphosphate (PI phosphat...

PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes

“…In line with previous studies (Perez et al, 2008), the lack of classical adherens junctions in human syncytiotrophoblast is likely to be associated with reduced Ecad dynamics and a weak PI3-K-dependent cytoskeletal rearrangement. Our results in human placental explants demonstrate that human cytotrophoblast Akt is slightly phosphorylated, whereas it is not detectable in the syncytiotrophoblast.…”

“…Perez et al (2008) proposed that in stable and cohesive epithelia, PI3-K activity is downregulated to allow the maintenance of Ecad interactions at cell-cell junctions, whereas in more dynamic epithelia, actin cytoskeleton remodeling requires PI3-K activity to disrupt adherens junctions and allow the formation of new sites of Ecad homophilic interactions. Our in vivo findings fit with this model: luminal accessibility of Ecad correlates with high PI3-K activity in intestinal GCs, ECs, and EEFs, where adherens junctions are dynamic (Madara et al, 1980;Madara and Trier, 1982;Kölsch et al, 2007;Marchiando et al, 2011).…”

Small Rho GTPase-mediated actin dynamics at endothelial adherens junctions

“…22 In line with this finding, a rapid increase in Rac1 activity upon VE-cadherin homotypic adhesion was detected biochemically, which was induced using beads coated with the VE-cadherin ectodomain. Thus, as was shown for E-, M-and N-cadherin, [64][65][66][67] we showed that VEcadherin can signal in an outside-in fashion to activate Rac1, providing a bidirectional feedback mechanism. 22 Of note, ongoing local remodeling of cell-cell junctions is observed even in apparently stable endothelial monolayers.…”

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…Alternatively, levels of activated Rac1 may eventually decrease at the contact site [30]. While a constitutively active form of Rac1 persists at cell contacts, a loss of both Rac1 and active Rac1 from newly formed MDCK cell contacts occurs within 5 min of E-cadherin recruitment [39]. RhoA activity would then increase and could result in membrane retraction via ROCK and myosin light chain (p-MLC) phosphorylation [40].…”