Adhesive and Lateral E-Cadherin Dimers Are Mediated by the Same Interface

Troyanovsky, Regina B.; Sokolov, Eugene P.; Troyanovsky, Sergey M.

doi:10.1128/mcb.23.22.7965-7972.2003

Cited by 75 publications

(91 citation statements)

References 34 publications

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“…The dissociation rate of strand-swapped dimers at cell-cell contacts in vivo may be slower than dissociation kinetics measured in vitro because in vivo dimers are likely reinforced by cis interactions (30,31). A similar Ca 2+ -independent stability has been described using cross-linking techniques for lateral dimers (32,33). Previous work examining cadherin dynamics (34) and the Ca 2+ dependence of cadherin interactions (35) demonstrated changes with a much longer latency (minutes or hours, rather than seconds) than we have observed here.…”

Section: Resultsmentioning

confidence: 86%

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Kim

Tai

Mok

et al. 2011

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

146

116

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Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherincadherin interactions are gated by extracellular Ca 2+ , which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s ), suggesting two types of native cadherin interactions-one that is rapidly modulated by changes in extracellular Ca 2+ and another with relatively stable adhesive activity that is Ca 2+ independent. The Ca 2+-sensitive dynamics of cadherin interactions were transmitted to the cell interior where β-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.cell adhesion | fluorescence resonance energy transfer | trans binding T he junctions between cells are populated with a variety of cell adhesion molecules that drive the recognition, assembly, and dynamics of cell-cell interactions. Parsing the distinct functions of different adhesion molecules has been challenging, in part due to a paucity of truly revealing in vivo assays. Among cell adhesion molecule families, the classic cadherins exhibit a unique dependence on extracellular Ca 2+ to rigidify the extracellular domains and enable trans junctional homophilic interactions. Three Ca 2+ ions bind with different affinities to each of the pockets between cadherin extracellular domains (1, 2). The affinity of the various Ca 2+ binding sites is in the micromolar to millimolar range (3-8), suggesting the possibility that cadherins can respond dynamically to changes in junctional Ca 2+ levels, and by virtue of physical interactions with cytoplasmic molecules, signal information about the junctional status to the cell interior.3D reconstructions of desmosomes by cryoelectron tomography reveal cadherins interact across intercellular interfaces (9, 10), and crystallographic data of several cadherin domains (4, 11, 12) implicate the first two extracellular repeats (ECs 1 and 2) as critical for homophilic interactions. Previous studies have indicated the importance of a highly conserved tryptophan residue (Trp2), present in the first EC domain, for cadherin-dependent adhesion (2, 12). This tryptophan inserts into the hydrophobic pocket of the partner N-cadherin (Ncad) molecule to form a strand-swapped dimeric structure (11, 12). Mutation of Trp2 to an alanine residue (W2A) prevents strand swapping (13) and results in a loss of adhesive function (2, 14, 15). Although extensive work has elucidated the structure (4, 9-14), biochemistry (2, 16) and single-molecule characterization (15, 17) of ...

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Section: Resultsmentioning

confidence: 86%

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Kim

Tai

Mok

et al. 2011

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

146

116

View full text Add to dashboard Cite

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“…All clones of human epidermoid carcinoma A-431 but Ec1M-C163A/V176C/ D155A-expressing clones were previously reported (Chitaev and Troyanovsky, 1998;Troyanovsky et al, 2003). New plasmids coding for this mutant were constructed using site-directed mutagenesis in the expression vector pRcCMV (Invitrogen, Carlsbad, CA).…”

Section: Cell Culture Dna Transfection and Plasmid Constructionmentioning

confidence: 99%

“…Cadherin sequences are numbered according to human E-cadherin (Bussemakers et al, 1993). Transfection, growth, immunofluorescence microscopy, and immunoprecipitation of the cells were done as described (Troyanovsky et al, 2003). In some experiments the actin filament inhibitors, cytochalasin D (Sigma, St. Louis, MO; final concentration 5 M) or latrunculin A (Molecular Probes, Eugene, OR; final concentration 0.2 M) were used.…”

Section: Cell Culture Dna Transfection and Plasmid Constructionmentioning

confidence: 99%

Stable and Unstable Cadherin Dimers: Mechanisms of Formation and Roles in Cell Adhesion

Troyanovsky

Laur

Troyanovsky

2007

MBoC

Self Cite

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Numerous attempts to elucidate the strength of cadherin dimerization that mediates intercellular adhesion have produced controversial and inconclusive results. To clarify this issue, we compared E-cadherin dimerization on the surface of living cells with how the same process unfolds on agarose beads. In both cases, dimerization was monitored by the same site-specific cross-linking assay, greatly simplifying data interpretation. We showed that on the agarose surface under physiological conditions, E-cadherin produced a weak dimer that immediately dissociated after the depletion of calcium ions. However, either at pH 5 or in the presence of cadmium ions, E-cadherin produced a strong dimer that was unable to dissociate upon calcium depletion. Both types of dimers were W156-dependent. Remarkably, only the strong dimer was found on the surface of living cells. We also showed that the intracellular cadherin region, the clustering of which through catenins had been proposed as stabilizer of weak intercadherin interactions, was not needed, in fact, for cadherin junction assembly. Taken together, our data present convincing evidence that cadherin adhesion is based on high-affinity cadherin-cadherin interactions.

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“…Although the cadherin cis interaction site was initially localized to the EC1 domains (10,24), it was later proposed that EC1 and EC2 domains of neighboring cadherin molecules are involved in these interactions (11,26). However, other studies using protein cross-linking and coimmunoprecipitation demonstrated that cis cadherin dimers were formed only in cells grown artificially at low calcium concentrations, and that cis and trans interactions shared the same adhesive interface (27)(28)(29). Although some electron tomography experiments have shown trans interactions involving both cadherin monomers and cis dimers (13), other cryoelectron tomographs suggest that cis dimerization is mandatory for trans adhesion (12).…”

mentioning

confidence: 99%

Resolving cadherin interactions and binding cooperativity at the single-molecule level

Zhang¹,

Sivasankar

Nelson

et al. 2009

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

179

195

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The cadherin family of Ca 2؉ -dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell-cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.atomic force microscope ͉ cell adhesion ͉ cis dimer ͉ fluorescence resonance energy transfer ͉ trans binding C adherins are Ca 2ϩ -dependent cell-cell adhesion proteins that play fundamental roles in the functional organization of cells and in maintaining the structural integrity of solid tissues (1, 2). Cadherin adhesion is modified during normal embryonic development, and disruption of adhesion is common in metastatic cancers (2-4). Despite detailed studies of cadherinmediated adhesion in multicellular organisms, a molecular understanding of the adhesive states of cadherin is less clear (5). Structural studies have shown that cadherin-cadherin contacts are mediated by the extracellular domain comprised of tandem repeats of 5 cadherin (EC) domains. A widely accepted model as summarized in textbooks (6) and recent review articles (1, 7-9) is that the functional unit of cadherin adhesion is a cis dimer formed by binding of the extracellular domains of 2 cadherins on the same cell surface. Cell-cell adhesion is initiated by the formation of trans adhesive complexes between the EC1 domains of cadherin cis dimers on opposing cell surfaces (1, 6-15). Strong cadherin adhesion may also require trans binding along additional EC domains (16)(17)(18)(19). Trans-cadherin binding is a lowaffinity interaction, but cell-cell adhesion is believed to be enhanced cooperatively by the lateral clustering of cadherins (20,21).Evidence for these trans-and cis-cadherin binding states, however, is controversial, and evidence for cooperativity in promoting adhesion is lacking. Models of cadherin cis dimerization are based on indirect evidence from the packing interactions in cadherin crystal structures (11,15,22,23), electron tomographs of desmosomes (12, 13), elect...

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Adhesive and Lateral E-Cadherin Dimers Are Mediated by the Same Interface

Cited by 75 publications

References 34 publications

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Stable and Unstable Cadherin Dimers: Mechanisms of Formation and Roles in Cell Adhesion

Resolving cadherin interactions and binding cooperativity at the single-molecule level

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