Untangling Desmosomal Knots with Electron Tomography

He, Weizhong; Cowin, Pamela; Stokes, David L.

doi:10.1126/science.1086957

Cited by 219 publications

(235 citation statements)

References 44 publications

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“…The structures of N-, E-, and C-cad- herin observed by crystallography and electron microscopy have shown end-and-end interactions that involve either EC1 or EC1-2 (4, 10 -18). Moreover, the recent interpretation of desmosomal cadherin interactions as revealed by electron microscopic tomography clearly reveals flexible tip-to-tip associations between the N termini of the molecules (17). Our findings are consonant with these studies, but in addition we have now demonstrated that the first two domains of N-cadherin are the minimal essential elements for the molecule to generate homophilic adhesion.…”

Section: Discussionsupporting

confidence: 79%

The Minimal Essential Unit for Cadherin-mediated Intercellular Adhesion Comprises Extracellular Domains 1 and 2

Shan

Yagita

Wang

et al. 2004

Journal of Biological Chemistry

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N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the "minimal essential unit" to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.Cadherins exist as a superfamily of calcium-dependent cell adhesion molecules. They play important roles in cell sorting in embryonic development and the maintenance of normal tissue architecture (1). The classical (type I) subfamily of cadherins (N-, E-, P-, and R-cadherin) comprises an extracellular domain (EC) 1 which consists of five similar tandemly arranged domains, a single pass transmembrane domain, and a conserved cytoplasmic segment (1). Homophilic cell-cell adhesion is mediated by the cadherin extracellular domains (2-4). The conserved cytoplasmic domain interacts with intracellular catenins that anchor cadherins to the cytoskeleton (5-9).Structural studies have revealed certain common features of classical cadherins that are likely responsible for adhesive engagement in the extracellular space (10 -17). The first model for N-cadherin activation derived from crystal structure analysis suggested that the first extracellular domain (EC1) forms cis-and trans-dimers and so contributes to N-cadherin homophilic associations (10, 13). It has also been shown that with increasing calcium concentration, the N-terminal domain of E-cadherin first forms a "ring-like" tip to tip lateral cis-dimer with an adjacent partner. Two cis-domains then interact ...

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Section: Discussionsupporting

confidence: 79%

The Minimal Essential Unit for Cadherin-mediated Intercellular Adhesion Comprises Extracellular Domains 1 and 2

Shan

Yagita

Wang

et al. 2004

Journal of Biological Chemistry

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“…Chemically-fixed or HPF/FSF intestinal samples were infiltrated, embedded, polymerized, sectioned and stained as previously described (He et al, 2003). Selected regions of 70 -200 nm sections were examined using an FEI T12 G2 Electron Microscope operating at 120 kV, and projection images were recorded on a Gatan 894 2K × 2K CCD camera (Gatan Corporation).…”

Section: Embedding and Electron Microscopymentioning

confidence: 99%

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

Kivork

Machinani

et al. 2007

Journal of Structural Biology

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We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically-fixed samples that reduced autonucleation, and a new pre-embedding goldenlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.

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“…Electron microscopy (12,13,24,31), X-ray crystallography (10,11,15,22), NMR (26,32), mutational experiments (22), and domain swapping experiments (33) have implicated the EC1 domain in cadherin trans interactions. However, direct force measurements (16)(17)(18) supported by cell attachment and bead binding assays (19) showed 3 distinct adhesive alignments interpreted as the overlap of opposing EC1-5, EC1-3, and EC1 domains.…”

mentioning

confidence: 99%

“…A widely accepted model as summarized in textbooks (6) and recent review articles (1,(7)(8)(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)(7)(8)(9)(10)(11)(12)(13)(14)(15). Strong cadherin adhesion may also require trans binding along additional EC domains (16)(17)(18)(19).…”

mentioning

confidence: 99%

“…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), electron micrographs of E-cadherin fusion constructs (24), and chemical cross-linking and gel filtration of cadherin extracted from cells (25). 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).…”

mentioning

confidence: 99%

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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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Untangling Desmosomal Knots with Electron Tomography

Cited by 219 publications

References 44 publications

The Minimal Essential Unit for Cadherin-mediated Intercellular Adhesion Comprises Extracellular Domains 1 and 2

The Minimal Essential Unit for Cadherin-mediated Intercellular Adhesion Comprises Extracellular Domains 1 and 2

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

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

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