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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., (© 2016 Rao and Zaidel-Bar. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-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 α-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 α-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 α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state., (Copyright © 2016 Biophysical Society. All rights reserved.)

E-cadherin is a trans-membrane tumor suppressor responsible for epithelial cell adhesion. E-cadherin forms adhesive clusters through combined extra-cellular cis- and trans-interactions and intracellular interaction with the actin cytoskeleton. Here we identify four populations of E-cadherin within cell junctions based on the molecular interactions which determine their mobility and adhesive properties. Adhesive and non-adhesive populations of E-cadherin each consist of mobile and immobile fractions. Up to half of the E-cadherin immobilized in cell junctions is non-adhesive. Incorporation of E-cadherin into functional adhesions require all three adhesive interactions, with deletion of any one resulting in loss of effective cell-cell adhesion. Interestingly, the only interaction which could independently slow the diffusion of E-cadherin was the tail-mediated intra-cellular interaction. The adhesive and non-adhesive mobile fractions of E-cadherin can be distinguished by their sensitivity to chemical cross-linking with adhesive clusters. Our data define the size, mobility, and adhesive properties of four distinct populations of E-cadherin within cell junctions, and support association with the actin cytoskeleton as the first step in adhesion formation., (© 2015. Published by The Company of Biologists Ltd.)

Actomyosin contractility is a highly regulated process that affects many fundamental biological processes in each and every cell in our body. In this Cell Science at a Glance article and the accompanying poster, we mined the literature and databases to map the contractome of non-muscle cells. Actomyosin contractility is involved in at least 49 distinct cellular functions that range from providing cell architecture to signal transduction and nuclear activity. Containing over 100 scaffolding and regulatory proteins, the contractome forms a highly complex network with more than 230 direct interactions between its components, 86 of them involving phosphorylation. Mapping these interactions, we identify the key regulatory pathways involved in the assembly of actomyosin structures and in activating myosin to produce contractile forces within non-muscle cells at the exact time and place necessary for cellular function., (© 2015. Published by The Company of Biologists Ltd.)

The ability to mechanically interact with the extracellular matrix is a fundamental feature of adherent eukaryotic cells. Cell-matrix adhesion in many cell types is mediated by protein complexes called focal adhesions (FAs). Recent progress in super resolution microscopy revealed FAs possess an internal organization, yet such methods do not enable observation of the formation and dynamics of their internal structure in living cells. Here, we combine structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to show that the proteins inside FA patches are distributed along elongated subunits, typically 300 ± 100 nm wide, separated by 400 ± 100 nm, and individually connected to actin cables. We further show that the formation and dynamics of these linear subunits are intimately linked to radial actin fiber formation and actomyosin contractility. We found FA growth to be the result of nucleation of new linear subunits and their coordinated elongation. Taken together, this study reveals that the basic units of mature focal adhesion are 300-nm-wide elongated, dynamic structures. We anticipate this ultrastructure to be relevant to investigation of the function of FAs and their behavior in response to mechanical stress., (© 2015 Wiley Periodicals, Inc.)

Background: Actomyosin contractility is the major cellular force driving changes in cell and tissue shape. A principal regulator of contractility is the small GTPase RhoA. External mechanical forces have been shown to impact RhoA activity and cellular contractility. However, the mechanotransduction pathway from external forces to actomyosin contractility is poorly understood., Results: Here, we show that actomyosin contractility in the C. elegans spermatheca is under control of RHO-1/RhoA, which, in turn, is regulated by the F-BAR and RhoGAP protein SPV-1. In the relaxed spermatheca, SPV-1 localizes through its F-BAR domain to the apical membrane, where it inhibits RHO-1/RhoA activity through its RhoGAP domain. Oocyte entry forces the spermatheca cells to stretch, and subsequently SPV-1 detaches from the membrane, permitting RHO-1 activity to increase. The increase in RHO-1 activity facilitates spermatheca contraction and expulsion of the newly fertilized embryo into the uterus, leading to relaxation of the spermatheca, SPV-1 membrane localization, and initiation of a new cycle., Conclusions: Our results demonstrate how transient membrane localization of a novel F-BAR domain, likely via specific binding to curved membranes, coupled to a RhoGAP domain, can provide feedback between a mechanical signal (membrane stretching) and actomyosin contractility. We anticipate this to be a widely utilized feedback mechanism used to balance actomyosin forces in the face of externally applied forces, as well as intrinsic processes involving cell deformation, from single-cell migration to tissue morphogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vertebrate adherens junctions mediate cell-cell adhesion via a "classical" cadherin-catenin "core" complex, which is associated with and regulated by a functional network of proteins, collectively named the cadherin adhesome ("cadhesome"). The most basal metazoans have been shown to conserve the cadherin-catenin "core", but little is known about the evolution of the cadhesome. Using a bioinformatics approach based on both sequence and structural analysis, we have traced the evolution of this larger network in 26 organisms, from the uni-cellular ancestors of metazoans, through basal metazoans, to vertebrates. Surprisingly, we show that approximately 70% of the cadhesome, including proteins with similarity to the catenins, predate metazoans. We found that the transition to multicellularity was accompanied by the appearance of a small number of adaptor proteins, and we show how these proteins may have helped to integrate pre-metazoan sub-networks via PDZ domain-peptide interactions. Finally, we found the increase in network complexity in higher metazoans to have been driven primarily by expansion of paralogs. In summary, our analysis helps to explain how the complex protein network associated with cadherin at adherens junctions first came together in the first metazoan and how it evolved into the even more complex mammalian cadhesome., (© 2014. Published by The Company of Biologists Ltd.)

Cell biologists studying cell adhesion have already figured out that cell-extracellular matrix connections, mediated by integrin receptors, are diverse and extremely complex structures. Dozens of adaptors-linking integrins with the cytoskeleton, and numerous enzymes and signaling proteins-regulating adhesion site dynamics, collectively referred to as the integrin adhesome, cooperate in mediating adhesion and activating specific signaling networks. Recent proteomic studies indicate that the known adhesome complexity is just the tip of the iceberg. In each existing category of molecular function the number of candidate components more than double the known components and several new categories are suggested. Proteomic analysis of different integrin heterodimers points to integrin-specific variations in composition and analysis of adhesion complexes under varying tension regimes highlights the force-dependent recruitment of different components, most notably LIM domain proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/beta-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Integrin-mediated adhesion is as ancient as multicellularity, but it was not always as complex as it is in humans. Here, I examine the extent of conservation of 192 adhesome proteins across the genomes of nine model organisms spanning one and a half billion years of evolution. The work reveals that Rho GTPases, lipid- and serine/threonine-kinases, and phosphatases existed before integrins, but tyrosine phosphorylation developed concomitant with integrins. The expansion of specific functional groups such as GAPs, GEFs, adaptors, and receptors is demonstrated, along with the expansion of specific protein domains, such as SH3, PH, SH2, CH, and LIM. Expansion is due to gene duplication and creation of families of paralogues. Apparently, these paralogues share few partners and create new sets of interactions, thus increasing specificity and the repertoire of integrin-mediated signaling. Interestingly, the average number of interactions positively correlates with the evolutionary age of proteins. While shedding light on the evolution of adhesome complexity, this analysis also highlights the relevance and creates a framework for studying integrin-mediated adhesion in simpler model organisms.