An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.

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Actin protrusions push on E-cadherin to maintain epithelial cell-cell adhesion

10.1101/644302 ◽

2019 ◽

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

AbstractCadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

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Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0429 ◽

2016 ◽

Vol 27 (18) ◽

pp. 2844-2856 ◽

Cited By ~ 31

Author(s):

Megha Vaman Rao ◽

Ronen Zaidel-Bar

Keyword(s):

Cell Adhesion ◽

Wound Repair ◽

Actin Polymerization ◽

Cell Junctions ◽

Epithelial Tissue ◽

Collective Cell Migration ◽

Epithelial Junctions ◽

Major Factors ◽

E Cadherin ◽

Cell Cell

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.

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A role for a micron-scale supramolecular myosin array in adherens junction cytoskeletal assembly

10.1101/2021.03.12.435158 ◽

2021 ◽

Author(s):

Hui-Chia Yu-Kemp ◽

Rachel A. Szymanski ◽

Nicole C. Gadda ◽

Madeline L. Lillich ◽

Mark Peifer

Keyword(s):

Epithelial Cells ◽

Cell Shape ◽

Shape Change ◽

Cell Junctions ◽

Actin Assembly ◽

Cell Shape Change ◽

Micrometer Scale ◽

Assembly Pathways ◽

E Cadherin ◽

Cell Cell

AbstractEpithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms used to build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized and highly organized actomyosin cytoskeleton at the zonula adherens, and combined genetic and pharmacological approaches with super-resolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micrometer-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, while associated actin filaments remained disorganized. This suggested these myosin arrays might bundle actin at mature junctions. Consistent with this, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization, and prevented actin bundling and polarization. These results suggest a novel mechanism by which myosin self-assembly helps drive actin organization to facilitate cell shape change.SummaryWe explored mechanisms epithelial cells use to assemble supramolecular actomyosin structures at E-Cadherin-based cell-cell junctions. Our data suggest individual actin assembly pathways are not essential. Instead, microscopy and pharmacological inhibition suggest micrometer-scale supramolecular myosin arrays help bundle actin at mature junctions.

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Actin protrusions push at apical junctions to maintain E-cadherin adhesion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908654117 ◽

2019 ◽

Vol 117 (1) ◽

pp. 432-438 ◽

Cited By ~ 12

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

Cadherin-mediated cell–cell adhesion is actin-dependent, but the precise role of actin in maintaining cell–cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell–cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell–cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

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HGF Converts ErbB2/Neu Epithelial Morphogenesis to Cell Invasion

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0567 ◽

2005 ◽

Vol 16 (2) ◽

pp. 550-561 ◽

Cited By ~ 77

Author(s):

Hanane Khoury ◽

Monica A. Naujokas ◽

Dongmei Zuo ◽

Veena Sangwan ◽

Melanie M. Frigault ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Cell Invasion ◽

Single Cells ◽

Epithelial Morphogenesis ◽

Cell Junctions ◽

Junction Protein ◽

Hepatocyte Growth ◽

E Cadherin ◽

Cell Cell

Activation of the hepatocyte growth factor receptor Met induces a morphogenic response and stimulates the formation of branching tubules by Madin-Darby canine kidney (MDCK) epithelial cells in three-dimensional cultures. A constitutively activated ErbB2/Neu receptor, NeuNT, promotes a similar invasive morphogenic program in MDCK cells. Because both receptors are expressed in breast epithelia, are associated with poor prognosis, and hepatocyte growth factor (HGF) is expressed in stroma, we examined the consequence of cooperation between these signals. We show that HGF disrupts NeuNT-induced epithelial morphogenesis, stimulating the breakdown of cell-cell junctions, dispersal, and invasion of single cells. This correlates with a decrease in junctional proteins claudin-1 and E-cadherin, in addition to the internalization of the tight junction protein ZO-1. HGF-induced invasion of NT-expressing cells is abrogated by pretreatment with a pharmacological inhibitor of the mitogen-activated protein kinase kinase (MEK) pathway, which restores E-cadherin and ZO-1 at cell-cell junctions, establishing the involvement of MEK-dependent pathways in this process. These results demonstrate that physiological signals downstream from the HGF/Met receptor synergize with ErbB2/Neu to enhance the malignant phenotype, promoting the breakdown of cell-cell junctions and enhanced cell invasion. This is particularly important for cancers where ErbB2/Neu is overexpressed and HGF is a physiological growth factor found in the stroma.

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Cortical Tension Initiates the Positive Feedback Loop Between E-cadherin and F-actin

10.1101/2021.02.23.432578 ◽

2021 ◽

Author(s):

Qilin Yu ◽

William R. Holmes ◽

Jean P. Thiery ◽

Rodney B. Luwor ◽

Vijay Rajagopal

Keyword(s):

Positive Feedback ◽

Feedback Loop ◽

Adherens Junctions ◽

Force Balance ◽

Intercellular Junction ◽

Cell Junctions ◽

Positive Feedback Loop ◽

Cortical Tension ◽

E Cadherin ◽

Cell Cell

AbstractAdherens junctions (AJs) physically link two cells at their contact interface via extracellular homophilic interactions between cadherin molecules and intracellular connections between cadherins and the actomyosin cortex. Both cadherin and actomyosin cytoskeletal dynamics are reciprocally regulated by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechano-chemical crosstalk that regulates AJ formation and homeostasis. The model couples a 2D lattice-based model of cadherin dynamics with a continuum, reaction-diffusion model of the reorganizing actomyosin network through its regulation by Rho signaling at the intercellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis less dependent manner. We further investigate how cadherin and actin regulate and cooperate. By considering the force balance during AJ maturation and the force-sensitive property of the cadherin/F-actin linking molecules, we show that cortical tension applied on the contact rim can explain the ring distribution of cadherin and F-actin on the cell-cell contact of the cell-doublet. Meanwhile, the positive feedback loop between cadherin and F-actin is necessary for maintenance of the ring. Different patterns of cadherin distribution can be observed as an emergent property of disturbances of this feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.Significance StatementThe formation, maintenance and disassembly of adherens junctions (AJs) is fundamental to organ development, tissue integrity as well as tissue function. E-cadherins and F-actin are two major players of the adherens junctions (AJs). Although it is well known that cadherins and F-actin affect each other, how these two players work together to maintain the intercellular contact is unclear. Using a novel mechano-chemical model of E-cadherin and F-actin remodeling, we demonstrate that a positive feedback loop between cadherins and F-actin allows them to stabilize each other locally. Mechanical and chemical stimuli applied to the cell adhesion change E-cadherin and F-actin distribution by consolidating or interrupting the feedback loop locally. Our study mechanistically links mechanical force to E-cadherin patterning at cell-cell junctions.

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Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells

Journal of Cell Science ◽

10.1242/jcs.111.9.1305 ◽

1998 ◽

Vol 111 (9) ◽

pp. 1305-1318 ◽

Cited By ~ 5

Author(s):

S.M. Norvell ◽

K.J. Green

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Extracellular Domain ◽

Full Length ◽

Intracellular Domain ◽

Epithelial Junctions ◽

Intracellular Domains ◽

E Cadherin ◽

Cell Cell

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing beta-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

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