E-cadherin interactome complexity and robustness resolved by quantitative proteomics

Guo, Zhenhuan; Neilson, Lisa J.; Zhong, Hang; Murray, Paul; Zanivan, Sara; Zaidel-Bar, Ronen

doi:10.1126/scisignal.2005473

Cited by 169 publications

(179 citation statements)

References 73 publications

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“…On Ecad-Fc-functionalized glass, cells were very flat and round, with large focal adhesion-like cadherin plaques at the end of thick radial F-actin bundles around the periphery ( Fig. 1E), similar to observations in previous studies (34)(35)(36)(37). These prominent F-actin and E-cadherin structures were very different from the organization of these proteins in cells adhered to Ecad-Fc PA gels.…”

Section: Resultssupporting

confidence: 78%

“…The elastic moduli of the two gel formulations used in this study were verified by atomic force microscopy (AFM): 10%T, 1%C, 27.5-31.8 kPa (median: 29.07 kPa) and 10%T, 2.5% C, 52.6-67.2 kPa (median: 57.34 kPa) (Table S1). We also prepared glass coverslips (with an elastic modulus of ∼100 GPa) coated with Ecad-Fc, which have been used in most other studies (34)(35)(36)(37).…”

Section: Resultsmentioning

confidence: 99%

“…Previous work showed that cells adhering to a more rigid (95-kPa) N-cadherin-coated PA gel were flat and well spread with large N-cadherin adhesion plaques (21), similar to cells adhered to Ecad-Fc-coated glass coverslips (∼100 GPa) (Fig. 1E) (34)(35)(36)(37). In contrast, cells adhered to a softer (10-kPa) N-cadherin-coated PA gel had a reduced spread area and smaller N-cadherin adhesions (21), similar to small E-cadherin clusters in cells adhering to 2D-supported membranes functionalized with the E-cadherin extracellular domain (58), 30-kPa and 60-kPa Ecad-Fc PA gels (Figs.…”

Section: Discussionmentioning

confidence: 99%

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Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of hightraction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42-and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cellcell adhesion.C ell adhesion is essential for tissue structure and function. Cells use specialized types of adhesions to interact with the surrounding environment, including integrin-based focal adhesions at cell-extracellular matrix (cell-ECM) contacts and cadherin-based adhesions at cell-cell contacts (1). Integrins bind to the ECM and intracellular proteins that link to the actin cytoskeleton and important signaling pathways (2). Similarly, cadherins regulate cellcell recognition and adhesion (3) and, through cytoplasmic adaptor proteins (catenins, vinculin) (4, 5), also link to the actin cytoskeleton and other proteins with signaling and scaffolding functions (6).Initiation of cell-cell adhesion requires significant reorganization of the actin cytoskeleton and is tightly controlled by the activities of actin nucleating proteins and Rho GTPases. Adhesion is initiated when filopodia from opposing cells come into contact with one another (7,8), and this process is regulated by Cdc42 activity (9, 10) and formin-dependent actin polymerization (11)(12)(13)(14). Intermediate stages of cell-cell contact formation involve lateral expansion of the contact by Rac1-induced and Arp2/3-dependent lamellipodial activity (15, 16). Finally, com...

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…E-cadherin is a multidomain protein consisting of five extracellular cadherin domains (ECD), a transmembrane domain, and a catenin-binding intracellular domain (ICD) (2)(3)(4). While the interaction between the E-cadherin ECDs from apposed cells physically holds the cells together, the E-cadherin-ICD interacts with the actin cytoskeleton by forming a tertiary complex with b-catenin and a-catenin (5)(6)(7)(8). The integration of E-cadherin ECD (E-cad-ECD) and E-cad-ICD-mediated interactions allows the mechanical coupling of adhering cells in the epithelial tissue and provides the potential for intercellular communication (9,10).…”

Section: Introductionmentioning

confidence: 99%

Sustained α -catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

Biswas

Hartman

Zaidel-Bar

et al. 2016

Biophysical Journal

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Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of a-catenin. Activation of a-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of a-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated a-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated a-catenin. Taken together, these results indicate that although the initial activation of a-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining a-catenin in the active state.

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“…In contrast, the Eph receptor ligand ephrin-A1 (EFNA1), which interacts with the extracellular domain of EphA2, was not present in the EphA2 interactome. Interestingly, our BioID analysis of primary keratinocytes did not identify E-cadherin, which was shown to be an EphA2 interactor in other epithelial cells (Van Itallie et al, 2014;Guo et al, 2014;Zantek et al, 1999). Taken together, the BioID analysis of the EphA2 interactome derived from primary human keratinocytes has greatly expanded the catalogue of putative proteins that may operate in concert with this RTK to regulate epithelial tissue homeostasis.…”

Section: +mentioning

confidence: 99%

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

White

Ventrella

Kaplan

et al. 2016

Journal of Cell Science

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EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions.

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E-cadherin interactome complexity and robustness resolved by quantitative proteomics

Cited by 169 publications

References 73 publications

Changes in E-cadherin rigidity sensing regulate cell adhesion

Changes in E-cadherin rigidity sensing regulate cell adhesion

Sustained α -catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

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