Molecular Mobility-Mediated Regulation of E-Cadherin Adhesion

Please cite this article in press as: Biswas, Molecular Mobility-Mediated Regulation of E-Cadherin Adhesion, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.012

Review

Molecular Mobility-Mediated Regulation of E-Cadherin Adhesion Kabir H. Biswas1,* Cells in epithelial tissues utilize homotypic E-cadherin interaction-mediated adhesions to both physically adhere to each other and sense the physical properties of their microenvironment, such as the presence of other cells in close vicinity or an alteration in the mechanical tension of the tissue. These position E-cadherin centrally in organogenesis and other processes, and its function is therefore tightly regulated through a variety of means including endocytosis and gene expression. How does membrane molecular mobility of E-cadherin, and thus membrane physical properties and associated actin cytoskeleton, impinges on the assembly of adhesive clusters and signaling is discussed.

Membrane-Localized Receptors Mediate Cellular Interaction Cells in a multicellular organism, such as a human being, express a variety of membrane-localized receptors that interact with their microenvironment including interstitial fluid, the extracellular matrix (see Glossary), and other neighboring cells. Although many of the receptors bind to freely diffusing ligands present in the interstitial fluid, including growth factor receptors such as epidermal growth factor receptor (EGFR) [1] and hormone receptors such as guanylyl cyclase C [2,3], others bind to ligands that are either present in the extracellular matrix, such as the integrins [4,5], or that are expressed on the membrane of neighboring cells, such as cadherins [6,7]. It has now become clear that, in addition to inducing allosteric conformational changes [8–14] or biochemical transformations, such as phosphorylation of the participating proteins [15], ligand binding-induced activation of these receptors also relays spatial and mechanical aspects of the microenvironment to downstream signaling events [5,16–22]. These latter aspects of cell signaling are specifically relevant to cell-adhesion receptors such as the integrins and cadherins. For instance, cellular application of mechanical force through integrins on physically fixed ligands, such as those displayed in the extracellular matrix by cell-borne receptors, results in activation of mechanical signaling in the cell, leading to profound changes in cell behavior [23–25]. Similar effects may be achieved between receptor–ligand pairs localized on the same cell membrane. This is enabled by the specific physicochemical properties of the cell membrane that is composed of different types of lipid molecules and proteins, and associates closely with the actin cortex and spectrin mesh. This gives rise to complex physical properties of the composite lipid bilayer and associated proteins, including an altered diffusion and assembly of nanoscale protein clusters and lipid domains [26–30]. Thus, in addition to serving as a platform for the assembly of cellular signaling complexes, the cell membrane directly impacts on receptor signal transduction. This article focuses on membrane molecular mobility-mediated regulation of a specific cadherin receptor, E-cadherin, and its role in adhesion assembly, as well as its possible implications in altered cellular conditions. First, this review briefly describes E-cadherins and the biochemical basis and mechanical regulation of E-cadherin adhesion. This is followed by a description of how these might relate to molecular mobility-mediated regulation of E-cadherin adhesion assembly. Recent studies have revealed that molecular diffusion of E-cadherin extracellular domains (ECDs) controls the frequency of E-cadherin cluster formation and, subsequently, adhesion-mediated mechanical signaling at these sites [31,32]. Combined analysis of epifluorescence images of the E-cadherin ECD on the bilayer and reflection interference contrast microscopy (RICM) images of cells forming adhesions on bilayers indicated that clustering of E-cadherin on the bilayer is coincident with the retraction of cellular filopodia. This led to the idea that molecular diffusion of E-cadherin on the bilayer impacts on its interactions with cell-borne E-cadherin, which in turn, regulates its clustering and mechanical

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Highlights E-cadherin is a key membranelocalized receptor that mediates adhesion in epithelial tissues and enables mechanical tension sensing. Molecular mobility of E-cadherin on a cell membrane includes both diffusive motion as well as active transport such as those mediated by the actin cytoskeleton. Reduced membrane molecular mobility is necessary for E-cadherin clustering and adhesion formation. E-cadherin clustering results in sustained activation of mechanical signal transducer a-catenin at adhesions. An alteration in membrane physical properties or a change in E-cadherin–actin cytoskeletal interaction may alter E-cadherin molecular mobility, thereby altering the assembly of E-cadherin-mediated cell–cell adhesion in the absence of any changes in the levels of the protein.

1College of Health and Life Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha 34110, Qatar

*Correspondence: [email protected]

https://doi.org/10.1016/j.tibs.2019.10.012 ª 2019 Elsevier Ltd. All rights reserved.

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Glossary

Extracellular domain

Cell exterior

Cell membrane β-Catenin Intracellular arr domain

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Cell interior F-actin Trends in Biochemical Sciences

Figure 1. E-Cadherin in the Cellular Context. A schematic representation of E-cadherin showing the cartoon of the extracellular domain (ECD) with Ca2+ ions indicated as dark blue dots, the transmembrane domain, and the intracellular domain (ICD). The E-cadherin ICD is complexed with b-catenin–a-catenin–F-actin (a-catenin in the ‘closed’ conformation). Also shown are other membrane proteins, some of which are glycosylated (gray hexagons), glycolipids and nanoscale lipid domains (blue lipids in the cell membrane).

signaling. Thus, an alteration in the molecular mobility of E-cadherin could impact on cell–cell adhesion formation, and thus crucially impact on cellular function in epithelial tissues.

E-Cadherin Mediates Cell–Cell Adhesion in Epithelial Tissues E-cadherin, a member of the classical calcium (Ca2+)-dependent cell-adhesion proteins, is a key component of intercellular cell–cell adhesions (adherens junctions) that are assembled between adjacent cells in epithelial tissues (Figure 1). Structurally, it comprises an ECD, a transmembrane domain, and an intracellular domain (ICD). The ECD can be subdivided into five domains (EC1–5) that are interspersed with four Ca2+ ion-binding sites [6]. EC1 domains in the apposing ECD interact in a homotypic fashion to mediate the adhesive function of E-cadherin. The ICD, on the other hand, is unstructured and interacts with the catenin family of proteins that connect E-cadherin to the actin cytoskeleton, a key component of cellular mechanical signaling that allows the generation and transmission of mechanical forces within and outside the cell. Thus, in addition to acting as an adhesive glue to physically keep cells together, Ecadherin functions to sense mechanical properties of the epithelial tissue [20], thereby providing control over processes both at the cellular level, such as cell polarity [33] and mechanical strain-induced cell cycle entry of quiescent cells [34], as well as at the tissue level, such as cell sorting [33] and contact inhibition of cell proliferation [35]. Overall, E-cadherin plays a fundamental role in tissue morphogenesis and homeostasis, and could also serve as a tumor suppressor [36,37]. The functional importance of E-cadherin is further increased by its interactions, either direct or indirect, with other membrane-associated molecular assemblies such as tight junctions [38], gap junctions [39], desmosomes [40], integrin-based adhesions [41], and pathogenic proteins such as botulinum neurotoxin A [42] and Listeria monocytogenes internalin [43]. Before we delve deeper into the molecular mobility-mediated regulation of E-cadherin adhesion, it is imperative to understand the key determinants of E-cadherin function and how they might impinge on molecular mobility-mediated regulation of E-cadherin adhesion.

Biochemical Regulation of E-Cadherin Adhesive Function The adhesive and signaling activities of E-cadherin are intricately regulated at multiple levels to shape cellular interactions in an organism. These include changes in its gene expression level (and thus protein levels in the cell), endocytosis-mediated regulation of its concentration on the cell membrane

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Actin cytoskeleton: filamentous actin (F-actin)-based molecular assemblies that play a crucial role in determining the shape and physical structure of a cell, and that enable the development of cellular tension and mechanical signaling. Adherens junction: also referred to as the zonula adherens or cell– cell junction, this is a molecular complex of E-cadherin and other proteins that forms at the interface of two apposing cells and connects the actin cytoskeleton in each of the adhering cells. Cadherins: a family of calciumdependent, membrane-spanning adhesion proteins that mediate physical adhesions of neighboring cells in a variety of tissues, including epithelial tissues, through their biochemical interactions. Catenins: a family of adaptor proteins, such as b- and a-catenin, that bind to cadherins and connect them to the actin cytoskeleton in adhering cells. Extracellular matrix: the organized meshwork of locally secreted biomaterial consisting of various proteins including fibrous proteins such as collagen that enable cell adhesion; it provides signals for cellular growth, migration, and differentiation. Filopodia: finger-like, thin, Factin-rich membrane protrusions that enable cells to probe their local environment. Integrins: a family of membranespanning receptors that typically bind to ligands in the extracellular matrix on the extracellular side and to the cellular cytoskeleton on the intracellular side enabling cellular adhesion and sensing of mechanical properties of the extracellular environment. Mechanical signaling: cellular signaling events that involve either the generation of mechanical tension through the cellular cytoskeleton and motor proteins or sensing of mechanical tension through structural alteration in signaling proteins. Molecular diffusion: the random motion of molecules such as proteins in a medium such as the cell membrane or the cytosol that is dictated by the kinetic energy of the molecule, and that is

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[44–48], biochemical interactions, and mechanical tension in the tissue [49]. This review focuses on the latter two aspects that do not involve any changes in protein levels of E-cadherin. First, Ca2+-dependent homotypic E-cadherin interactions (trans-interactions) proceed via the formation of a weakly interacting intermediate, an X-dimer structure, upon initial encounter of monomers [50–53]. Not only does it form the basis for initial interaction but the X-dimer intermediate also provides a mode for dimer dissociation [54], thus providing a mechanistic basis for the reversibility of Ecadherin homodimerization. The weakly interacting X-dimer intermediate matures into a stable homodimer with the formation of additional interactions [50]. These trans-interacting homodimers can further interact in cis (cis-interaction), leading to the formation of a highly dense molecular array of E-cadherin at adhesion sites [50,51,55]. Additional modes of regulation of E-cadherin homodimerization include allosteric regulation by the ICD [9,56] and differential glycosylation of the ECD [57– 60].

dependent on its molecular properties, such as its mass, and on the properties of the medium, such as its viscosity. Molecular mobility: the cumulative motion of molecules such as proteins that includes both pure diffusion and active transport by cellular processes.

E-cadherin ECD homodimerization is directly coupled to the cellular cytoskeleton through the ICD that interacts with a large of number of intracellular proteins [18,61–63]. These include a direct interaction with p120 catenin and b-catenin. The latter, in turn, interacts with a-catenin, a multidomain adaptor protein containing an actin-binding domain, thus serving as a bridge between E-cadherin and the actin cytoskeleton [64]. In addition, the E-cadherin ICD undergoes post-translational modifications [65] such as phosphorylation, which could impact on its interaction with the catenin family of proteins [15,66], and ubiquitinylation, which leads to a reduction in the cell-surface expression of E-cadherin through endocytosis [45,67].

Mechanical Signaling at E-Cadherin Adhesions The physical linkage of the actin cytoskeleton of adhering cells through E-cadherin raises the possibility of mechanically regulated cellular signaling [34,68,69], wherein actomyosin-generated tension in cells leads to changes in the biochemical and/or biophysical properties of E-cadherin [34,70–72]. Indeed, this has been shown to be the case both at the extracellular as well as the intracellular side. At the extracellular side, the X-dimer intermediate has been shown to exhibit catch-bond behavior, wherein the time required for dissociation of the interacting monomers (bond life-time) increases with an increase in externally applied tensile forces mimicking actomyosin-generated mechanical tension on the monomers [73,74]. Such an increase in bond lifetime peaks at a force of 30 picoNewtons, followed by a decrease in the lifetime with a further increase in applied forces, indicating a transition of the interaction to regular slip-bond behavior. These measurements arise from single-molecule force-clamp experiments performed with the W2A mutant E-cadherin that can only form the X-dimer intermediate structure but cannot go on to form the mature strand-swapped dimer. In addition, the force-dependent catch-bond behavior of E-cadherin requires a saturating concentration of Ca2+ ions because a reduction in the Ca2+ concentration results in the loss of catch-bond behavior, although it could still interact homotypically [74]. Importantly, these experiments also revealed an unusual behavior of E-cadherin dimers in that they require a relatively prolonged interaction time (3 s) to form the stable, strand-swapped structure that displays slip-bond behavior. However, they displayed an ideal bond behavior wherein the dissociation lifetime, although short, was independent of the externally applied force when the E-cadherin monomers were allowed to interact with each other for shorter period (0.3 s). These findings suggest that conversion of the X-dimer intermediate to the stable strand-swapped dimer is a relatively slow process. At the intracellular side, an increase in the mechanical tension in the tissue results in the reinforcement of E-cadherin adhesion, enabling it to withstand physical changes in the tissue. This appears to occur via an increased recruitment of the a-catenin interacting protein, vinculin, to E-cadherin adhesions [75,76]. Akin to its role in mechanical signal transduction at integrin-based adhesions, increased recruitment of vinculin, which is homologous to a-catenin and contains an actin-binding domain [77], enables E-cadherin adhesion to strengthen its interaction with the actin cytoskeleton. The key molecular event in this process involves a force-induced conformational change in a-catenin from a ‘closed, inactive’ to an ‘open, active’ structure [32,63,64,78,79], making cryptic binding sites

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accessible to vinculin and leading to a large increase in the affinity of vinculin for a-catenin, as seen in single-molecule force spectroscopy experiments [80]. It is important to note that the direct interaction between a-catenin and the actin cytoskeleton is also sensitive to forces applied to the actin filaments, as revealed by optical trap-based, molecular reconstitution experiments [79]. Analysis of homodimeric a-catenin structure, in conjunction with that of vinculin, raise the possibility that, when the a-catenin is bound to b-catenin and the E-cadherin ICD, the probable F-actin binding domain in a-catenin may not be available for F-actin binding [81]. This provides a mechanistic basis for the lack of interaction between the b-catenin–a-catenin complex and F-actin in solution [82]. In addition, a recent report using a variety of approaches, including structural and in vivo experiments, elucidated the mechanism by which parts of a-catenin allosterically interact with F-actin [13]. This raises an important question regarding the basis of initial interactions between a-catenin and F-actin that allow application of increased tension to the E-cadherin–b-catenin–a-catenin complex. However, live-cell experiments using a fluorescence resonance energy transfer (FRET)-based E-cadherin tension sensors indicate that E-cadherin is constitutively under actomyosin tension in cells [83], suggesting that the E-cadherin–b-catenin–a-catenin complex interacts constitutively with the cellular actin cytoskeleton. It appears plausible that a fraction of a-catenin in complex with E-cadherin and b-catenin is present in a conformation that allows it to interact with F-actin, an idea supported by recent in vitro experiments [11,84], and an increase in actomyosin tension allows an enhanced interaction between a-catenin and F-actin [64].

Diffusion of E-Cadherin on Synthetic Membranes Regulates Adhesion Assembly Cell-based assays have shown that E-cadherin is enriched at the cell–cell interface in the form of micron-scale clusters [31,32,85]. This is primarily caused by trans-interactions between E-cadherin molecules from apposing cells, although cis-interactions between molecules in the same cell may provide an additional drive towards it [51]. These are phenomenologically similar to the enrichment of receptor–ligand complexes at the interface formed between vesicles and planar bilayers, both in theory [86] as well as in experiments [87]. These micron-scale clusters have been further resolved by super-resolution microscopy to consist of nanoscale clusters that have molecular densities close to those seen in crystals [48,55,88]. The sizes of these nanoscale clusters were, however, found to be polydisperse [48]. In addition, E-cadherin density in the nanoscale clusters is dependent on cis-interactions between the ECDs, and the size of the clusters is regulated by the underlying actin cytoskeleton through the ICD [88]. The nanoscale organization of specific lipid species in the cell membrane may further promote local clustering of E-cadherin [89,90], as well as an interaction between the transmembrane domains [91]. Importantly, relatively smaller nanoscale clusters of E-cadherin were observed even in the absence of homodimerization, such as those present at non-adhering cell interfaces or in ECD-deleted transmembrane and ICD-containing protein constructs, leading to the proposition that these smaller nanoscale clusters are the building blocks of E-cadherin adhesion [7,88]. Although individual E-cadherin molecules form homotypic interactions in solution with low affinity, the assembly of E-cadherin adhesion in a wide variety of experimental model systems, such as cells in culture or in organisms such as nematode, fly, or mouse embryos, appears to be an active process involving F-actin-based cellular structures such as filopodia [7,92–100]. This became apparent in reconstitution experiments using a hybrid live cell-supported lipid bilayer platform (Figure 2) [21,101]. In this set-up, a live cell expressing a receptor of interest is allowed to interact with a biointerface consisting of a ligand-functionalized synthetic lipid bilayer assembled on a solid support [101] to microscopically monitor the spatial and temporal evolution of receptor–ligand complexes on the cell membrane [19,21,102,103]. This system allows straightforward manipulation of various features of the membrane, including ligand identity and density, or its physical properties, such as viscosity, as well as the creation of nano- and microscopic features for restricting free movement of receptor–ligand complexes on the supported lipid bilayer. Epithelial cells interacting with supported

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Figure 2. Membrane Diffusion-Regulated E-Cadherin Adhesion Formation. (A) A schematic representation of an E-cadherin molecule on a fluid (left) versus a viscous (right) supported lipid bilayer. The E-cadherin molecule on the fluid bilayer diffuses rapidly whereas it diffuses slowly on a viscous bilayer. (B) FRAP (fluorescence recovery after photobleaching) analysis of fluorescently labeled E-cadherin-ECD on a fluid (left) versus a viscous (right) supported lipid bilayer. Graphs in the lower panel show fluorescence intensity line scans before and after photobleaching of a fluid and a viscous bilayer. Note that the fluid bilayer shows a rapid recovery of fluorescence signal while the viscous bilayer does not show recovery even after 30 minutes of photobleaching of a similarly sizedarea on the bilayer. (C) Epifluorescence images of fluorescently labeled E-cadherin on a fluid (left) versus a viscous (right) supported lipid bilayer showing the formation of a compact zone of enrichment on a fluid bilayer but a ring-like zone of enrichment on a viscous bilayer. The color bar indicates the increase in the local E-cadherin fluorescence intensity relative to the bulk of the bilayer. Scale bar, 5 mm. (D) Graph showing the percentage of cells showing enrichment of E-cadherin-ECD on fluid (left) versus viscous (right) supported lipid bilayers. Data represent means G SD obtained from multiple experiments. Panels (B–D) are modified, with permission, from [31].

membrane functionalized with the E-cadherin ECD show extensive filopodial extension and retraction, and E-cadherin clustering is associated with retracting filopodia, which is abolished upon pharmacological inhibition of filopodia formation [31]. Clustering is an actomyosin tension-dependent process because treatment of cells with blebbistatin, an inhibitor of force-generating myosin II,

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results in a loss of adhesion formation [31]. In addition, adhesion formation appears to be an active process requiring cellular energy because inhibition of cellular ATP-generating proteins results in a loss of adhesion formation [31]. One key finding emerging from these reconstitution experiments is the role of the molecular mobility of E-cadherin in adhesion assembly. It appears that filopodia-borne E-cadherin molecules initially interact with bilayer-bound E-cadherin ECD to form low-affinity X-dimer intermediates [50–53]. These weakly interacting intermediate structures transform into stable, strand-swapped homodimers and form clusters while the cell is retracting the filopodia. This transition, however, is sensitive to the molecular mobility of the bilayer-bound E-cadherin ECD [31]. Thus, E-cadherin ECDs displayed on viscous bilayers possessing low diffusive mobility are frequently able to assemble adhesions, whereas those on fluid bilayers possessing high diffusive mobility (D of 1.6 G 0.2 mm2/s) often fail to do so. It is important to note that the low percentage of cells that go on to assemble E-cadherin adhesions show complete adhesion formation (i.e., an all-or-none phenomenon) [31]. This observation led to the idea that cadherin adhesion formation involves a step of kinetic nucleation, and thus reduced molecular mobility of the E-cadherin ECD displayed on viscous bilayers increases the chances of nucleation and clustering during filopodia retraction. On the other hand, highly diffusive proteins displayed on fluid bilayers dissipate into the bulk, with a concomitant decrease in the chances of nucleation and clustering during filopodia retraction. It is also possible that increased viscous drag, which is expected to appear on viscous bilayers but not on fluid bilayers, increases the mechanical tension between the weakly interacting monomers in the X-dimer intermediate [50–53]. This would result in an increase in the lifetime of the X-dimer catchbond [73,74], leading to an increase in the probability of strand-swapped dimer formation. Theoretical estimation of the force generated on the bilayers using the Einstein Smoluchowski relation suggests that the diffusion coefficient of the bilayer-bound E-cadherin molecule must be low (high viscosity) to achieve the range of forces that are required for the increased lifetime of the X-dimer catch-bond [31], and thus for an increased frequency of nucleation and clustering of E-cadherin at the cell–bilayer interface. Either alone or in combination, both effects (i.e., increased nucleation and X-dimer catch-bond lifetime) of reduced diffusion of the E-cadherin ECD on the bilayer would increase the frequency of nucleation and clustering of E-cadherin, and thus of adhesion formation in the hybrid setup. This nucleation and clustering of E-cadherin is, however, not dependent on cis-interactions because mutation of the cis-interaction interface does not significantly impact on adhesion formation on viscous bilayers [31]. The E-cadherin ICD is essential for cell–cell adhesion formation because its deletion results in loss of adhesion formation, thus indicating a crucial role for E-cadherin–actin cytoskeleton interactions in adhesion formation [7,31]. Although this interaction is required, it is not sufficient for adhesion formation on the bilayers because direct fusion of the actin-binding domain of a-catenin to the E-cadherin ICD does not alleviate the requirement for low E-cadherin bilayer diffusion for adhesion formation. Similarly, the presence of physical barriers on the supported lipid bilayer substrate [21] does not increase the percentage of cells forming adhesions, even though they impact on a-catenin conformational activation [32]. It is possible that clustering increases the tension on E-cadherin molecules, resulting in an increase in the strength of the intermediate X-dimer interaction [73,74]. This eventually would lead to an increase in the probability of strand-swapped dimer formation, and thus of adhesion assembly. Indeed, hybrid live cell-supported lipid bilayer experiments using nanopatterned substrates indicate that the conformational ‘opening’ of a-catenin, as assessed by the binding of a conformationally sensitive antibody [78–80], which is a signature of mechanical force application to E-cadherin clusters, is dependent on the nucleation and micron-scale clustering of E-cadherin [32]. In fact, once ‘open’, a-catenin does not revert back to the ‘closed’ conformation immediately after a decrease in the cellular actomyosin tension, as seen in other experiments [78,104]. This perhaps reflects a more complex regulation of

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a-catenin structure that explores multiple conformations [63,64] that are likely influenced by phosphorylation [105] and interactions with other cadhesome proteins [61,62]. A reduction in the molecular mobility of a protein is generally thought to decrease the probability of its interaction with other proteins by decreasing the probability of their encounter. In agreement, a reduction in molecular mobility, such as in the case of the T cell receptor [106] or the presence of barriers on the cell membrane such as those seen in the case of phagocytic receptor CD44 [107], negatively impacts on receptor function. In addition, no other receptor studied using the hybrid live cell-supported lipid bilayer setup, such as the T cell receptor, the EphA2 receptor tyrosine kinase, or integrin adhesion [17,21], showed a dependence on reduced ligand molecular mobility, although each of these receptor systems has mechanical force-sensing built into it. Therefore, E-cadherin adhesion appears to be unique in terms of its requirement for reduced molecular diffusion.

E-Cadherin Mobility on the Cell Membrane Can Be Reduced in Multiple Ways The observation of diffusion-dependent E-cadherin adhesion formation on synthetic supported lipid bilayers is interesting but certainly raises the question as to how it might be relevant in an in vivo context. The fluid synthetic supported lipid bilayers used in the reconstitution experiments are assembled on a solid support with a relatively simple lipid composition displaying a single physical phase, in other words this is a liquid disordered phase. Thus, the E-cadherin ECD displayed on such bilayers exhibits molecular mobility that largely reflects the diffusion of anchoring lipid molecules, as assessed by both fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) analyses [21,31]. However, this is far from the molecular mobility of the protein on actual cell membranes (Figure 1). First, cell membranes are composed of multiple types of lipid molecules that possess distinct physicochemical properties, and thus may exist in different phases. Some of these lipid types can form local aggregates in the form of nanodomains [108]. Second, the presence of other proteins and the glycocalyx on cell membranes further increases their complexity, leading to significant changes in their physical properties in comparison to simple synthetic bilayers [26–30]. Further, the presence of cytoskeletal elements in close proximity to the cell membrane can impede free diffusion of any protein on the cell membrane [109,110]. Thus, although the physical organization and biophysical properties of cell membranes are still debated, it is clear that the molecular mobility of proteins associated with cell membranes is generally lower than that seen on synthetic membranes. Thus, membrane-borne E-cadherin molecules are expected to experience a general reduction in their molecular mobility. In addition to these general effects, the interaction of E-cadherin ICD with the underlying cellular actin cytoskeleton or spectrin mesh will cause a further decrease in its molecular mobility to below that on a synthetic bilayer [18,20,33,78,79,83,111]. In fact, live-cell single-particle tracking experiments have revealed different types of mobility behaviors of E-cadherin, including free diffusion and restricted, corralled movements [112,113]. In addition, single-molecule tracking [112,113] and super-resolution imaging studies [48,88] have shown that the presence of nanoscale clusters of E-cadherin will certainly reduce the molecular mobility of E-cadherin on the cell membrane. A fluorescence recovery after photobleaching (FRAP)-based report suggest that E-cadherin could be immobilized on the cell membrane even in the absence of adhesive interactions through the ECD [114]. Thus, a fraction, at least, of E-cadherin expressed on the cell membrane possess low molecular mobility, as seen on viscous supported lipid bilayers, and is thus poised for adhesion formation.

Concluding Remarks Reconstitution of E-cadherin adhesion in a hybrid format using synthetic supported lipid bilayers and live cells has provided novel insights into its regulation that could not have been gained from either in vitro biochemical and biophysical assays or purely cell-based assays. Based on these results, it is possible to propose that cells interrogate their microenvironment using E-cadherin borne on Factin-based processes such as filopodia (Figure 3) [7,31]. The dependence of E-cadherin on its diffusion on the supported lipid bilayer suggests that an increase in its molecular mobility on the cell

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Outstanding Questions How do cells utilize molecular mobility of E-cadherin in regulating their adhesion to other cells? How does molecular mobility of Ecadherin regulate its interaction with other proteins that are known to associate with it? Are there other receptors that require a reduced molecular mobility to be functional?

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Figure 3. E-Cadherin Adhesion Assembly. A schematic showing various plausible steps in the assembly of E-cadherin adhesions. Initial encounter between Ecadherin monomers from apposing cells results in the formation of an intermediate (X-dimer) E-cadherin homodimer [50–53]. This intermediate then transforms into a stable, strand-swapped homodimer and cluster depending on the molecular mobility of E-cadherin: in other words, E-cadherin possessing low molecular mobility has a higher probability of nucleating a cluster, whereas E-cadherin with high molecular mobility often fails to nucleate a cluster [7,31]. Once clusters of strand-swapped dimers are formed, a-catenin is conformationally activated from a ‘closed’ form to an ‘open’ form, likely due to increased actomyosin tension [7,64].

membrane in vivo could abrogate its ability to form adhesions. This could happen under circumstances where there are changes in the cellular cytoskeleton [22,79,83,88,94,115] or a change in the lipid composition of the cell membrane [89,90]. Other probable factors that might impact on E-cadherin molecular mobility include an alteration in the local Ca2+ ion concentration, which impacts on both its molecular mobility as well as on catch-bond behavior [74,116], or an alteration in its glycosylation pattern [57–60]. Thus, even before a cell loses expression of E-cadherin via transcriptional regulation, as seen in processes such as epithelial to mesenchymal transition, E-cadherin may be rendered incapable of cellular adhesion because of an increase in its molecular mobility. In addition, although these studies have primarily focused on the assembly of E-cadherin adhesion, it remains to be seen whether its molecular mobility impacts on its interaction with other membrane-localized receptors such as tight junction proteins [38], integrins [41], or toxins that function by disrupting E-cadherin clusters [42] (see Outstanding Questions).

Acknowledgments This work was supported by the College of Health and Life Sciences, Hamad Bin Khalifa University, a member of the Qatar Foundation.

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