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Actin Protrusions Push at Apical Junctions to Maintain E-Cadherin Adhesion

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Actin Protrusions Push at Apical Junctions to Maintain E-Cadherin Adhesion Actin protrusions push at apical junctions to maintain E-cadherin adhesion John Xiao He Lia, Vivian W. Tanga, and William M. Briehera,1 aDepartment of Cell and Developmental Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Janet Rossant, Hospital for Sick Children, University of Toronto, Toronto, Canada, and approved November 20, 2019 (received for review May 19, 2019) Cadherin-mediated cell–cell adhesion is actin-dependent, but the safety mechanisms to ensure that important functions remain ro- precise role of actin in maintaining cell–cell adhesion is not fully bust in the face of a perturbation or occasional failure. understood. Actin polymerization-dependent protrusive activity is We thus reasoned that actin polymerization-dependent pro- required to push distally separated cells close enough to initiate trusive activity might operate as a safety mechanism to keep contact. Whether protrusive activity is required to maintain adhe- lateral membranes of neighboring cells close to each other to sion in confluent sheets of epithelial cells is not known. By electron promote E-cadherin binding. Protrusions are known to be im- microscopy as well as live cell imaging, we have identified a pop- portant for initiating the formation of cell–cell adhesion at the ulation of protruding actin microspikes that operate continuously leading edge of motile cells (24, 25). However, cell–cell adhesion near apical junctions of polarized Madin-Darby canine kidney in mature epithelial sheets (7, 8, 26–28) is intrinsically more (MDCK) cells. Live imaging shows that microspikes containing E- stable than that in newly forming epithelial monolayers (24, 25). cadherin extend into gaps between E-cadherin clusters on neigh- Previous work in endothelial cells has shown lamellipodium- and boring cells, while reformation of cadherin clusters across the cell– filopodium-like protrusions at the junctions of motile cells and cell boundary correlates with microspike withdrawal. We identify newly forming monolayers (29–33). At mature endothelial Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for junctions, lamellipodium-like protrusions crawl over the top of microspike formation. Depleting these factors from cells using RNA the neighboring cell at the sites lacking vascular endothelial interference (RNAi) results in myosin II-dependent unzipping of (VE)–cadherin clusters, so that VE–cadherin clusters can form cadherin adhesive bonds. Therefore, actin polymerization-dependent at the new cell–cell contact created by the lamellipodium and protrusive activity operates continuously at cadherin cell–cell junctions integrate into and strengthen the linear junction when the to keep them shut and to prevent myosin II-dependent contractility lamellipodium retracts (31). At unstable or remodeling en- from tearing cadherin adhesive contacts apart. dothelial junctions, bundled actin structures from 1 cell are engulfed by the neighboring cell and associated with VE– E-cadherin | actin | epithelial | junction | adhesion cadherin, although they are thought to enhance adhesion through filopodial retraction (19, 29, 30, 34). Above all, whether adherin family cell–cell adhesion molecules are essential for protrusive activity continues to operate in mature epithelial junc- Ctissue cohesion and organization throughout life. While the tions is not known. Junctional actin assembly in epithelia depends cadherin ectodomain mediates homophilic binding, strong ad- on factors associated with protrusive actin networks (2, 3, 23, 35). hesion requires contributions from cytosolic factors including the Thus, we looked for protrusive activities in Madin-Darby canine actin cytoskeleton (1). The apical junction of cell–cell interface is kidney (MDCK) (kidney tubular epithelial) cell sheets 3–4 the prominent site of actin polymerization in epithelial cells even d postconfluency with established apical–basal polarity. long after the junction has been established (2–4). However, we still do not fully understand either the function or the regulation Significance of the actin at cell–cell adhesive junctions. Cells generally organize actin into either contractile networks All solid tissues rely on cadherin family cell–cell adhesion mole- that use myosin to generate pulling forces or protrusive networks cules for their cohesion and organization. Cadherin adhesive that use actin polymerization to generate pushing forces (5). function depends on the actin cytoskeleton, but actin’s contribu- Actomyosin contractility plays a major role in cadherin biology, tion to cell–cell adhesion is not fully understood. We demonstrate especially during development when cadherin adhesive junctions that actin polymerization-dependent protrusive activity operates propagate tensile forces across interconnected sheets of cells to continuously to push lateral membranes of neighboring cells to- drive various cell movements (6). Contractility also contributes gether to keep cadherins in contact. Actin-dependent protrusive to junction maturation and stabilization in epithelial sheets (7, 8). activity functions as a safety mechanism to quickly repair cadherin Junctional actin polymerization is suggested to build contractile adhesive junctions whenever they fail and to prevent myosin- actomyosin (9–14). Observations in cells strongly suggest that dependent contractile forces from tearing the junctions further apart. Since loss of cadherin adhesion is associated with a number cadherin–catenin complexes couple to contractile actin networks of diseases including cancer progression, then misregulation of and that the complex is under tension (8, 15, 16). actin protrusive activity should be considered as a possible con- Coupling cadherins to the contractile actin cytoskeleton offers tributing factor to epithelial pathophysiology. great morphogenetic power to sculpt tissues, but relying on only contractile forces to stabilize junctions in established epithelia is Author contributions: J.X.H.L., V.W.T., and W.M.B. designed research; J.X.H.L. and V.W.T. not fail-safe. In vitro measurements show that piconewton pull- performed research; J.X.H.L. analyzed data; and J.X.H.L. and W.M.B. wrote the paper. ing forces stabilize the connection between the cadherin–catenin The authors declare no competing interest. complex and F-actin (17). In addition, myosin-dependent con- This article is a PNAS Direct Submission. tractility leads to adherens junction remodeling, perhaps to fortify Published under the PNAS license. the junction and make it resilient against tearing (7, 11, 18, 19). 1To whom correspondence may be addressed. Email: [email protected]. However, continuing to pull on a broken junction would only tend This article contains supporting information online at https://www.pnas.org/lookup/suppl/ to propagate the defect, which could tear tissues apart (20–23). doi:10.1073/pnas.1908654117/-/DCSupplemental. Cadherin-mediated adhesion is important, and cells tend to evolve First published December 23, 2019. 432–438 | PNAS | January 7, 2020 | vol. 117 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1908654117 Downloaded by guest on October 1, 2021 − − Results rate of 1.1 μmmin 1 and −1.1 μmmin 1, respectively (Fig. 1F). By thin-section electron microscopy we found membrane protru- After falling back into the cell body, some microspikes bundle with sions of 389 ± 21 nm in length (mean ± SEM, n = 20 microspikes) the junctional actin belt (SI Appendix,Fig.S1C). The frequency that burrowed into neighboring cells near the apical junctional of actin microspikes is 0.53 ± 0.05 per μm of junction length per complex in MDCK cells (Fig. 1 A and B and SI Appendix,Fig. minute (mean ± SEM, n = 20 cells). Membrane-targeted yel- S1A). Forty-one percent of junctions (n = 66) in MDCK cells have low fluorescent protein (YFP) also showed dynamic protru- protrusions. We also examined Caco-2 intestinal epithelial cells sions (Fig. 1G, SI Appendix,Fig.S1E,andMovie S2). The − − (C2bbE1 clone) and found protrusions at 41% of junctions (n = frequency of membrane protrusions is 0.41 ± 0.04 μm 1 min 1 51) (Fig. 1C and SI Appendix,Fig.S1A). Lateral membrane pro- (mean ± SEM, n = 21 junctions in 5 cells). Blocking actin- trusions have long been recognized (3, 36), but we found protru- filament (+) end dynamics with cytochalasin D (41) elimi- sions also prevalent near apical junctional complex which consists nated microspikes, indicating their dependence on actin as- of tight junctions and adherens junctions (1). The intercellular sembly (Fig. 1H). Simultaneous imaging of actin and E- distance between the tip of a protrusion and the neighboring cell’s cadherin and kymograph analysis showed E-cadherin tracks membrane is 20–40 nm, resembling that between adherens junc- along 88% of elongating microspikes (n = 33) and 86% of tions (Fig. 1B)(37–39). Using live cell imaging of F-actin marker retracting microspikes (n = 21), respectively (Fig. 1E and SI UtrCH, we discovered similar filopodium-like microspikes (Fig. Appendix,Fig.S1D). 1D) (40). By examining a single labeled cell in a cell sheet, we were We next asked which actin assembly factors promote micro- able to see membrane structures that are otherwise masked by spikes. Previous work identified EVL, CRMP-1, and Arp2/3 as 3 homogenous labeling, like immunofluorescence. Actin micro- factors necessary for actin assembly at apical cell–cell junctions spikes are 381 ± 18 nm long and persist for 6 ± 1 s (median ± (2, 3, 23). Arp2/3 nucleates the formation of new actin filaments
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