Direct molecular force measurements of multiple adhesive interactions between cadherin ectodomains

Sivasankar, Sanjeevi; Brieher, William M.; Lavrik, Nickolay V.; Gumbiner, Barry M.; Leckband, Deborah

doi:10.1073/pnas.96.21.11820

Cited by 160 publications

(173 citation statements)

References 35 publications

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“…The soluble, recombinant extracellular domain EC1-5 of Xenopus cleavage stage cadherin with a C-terminal hexahistidine tag was stably expressed in Chinese Hamster Ovary cells (27,28). The soluble, expressed hexahistidinetagged ectodomains were first purified by affinity chromatography on an Affi-Gel column (Bio-Rad, Hercules, CA), as described in Sivasankar et al (28).…”

Section: Methodsmentioning

confidence: 99%

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Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

Cai

Shashikanth

Leckband

et al. 2016

Biophysical Journal

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Ca 2þ ions are critical to cadherin ectodomain rigidity, which is required for the activation of adhesive functions. Therefore, changes in Ca 2þ concentration, both in vivo and in vitro, can affect cadherin conformation and function. We employed single-molecule tracking to measure the diffusion of cadherin ectodomains tethered to supported lipid bilayers at varying Ca 2þ concentrations. At a relatively high Ca 2þ concentration of 2 mM, cadherin molecules exhibited a fast diffusion coefficient that was identical to that of individual lipid molecules in the bilayer (D fast z 3 mm 2 /s). At lower Ca 2þ concentrations, where cadherin molecules were less rigid, the ensemble-average cadherin diffusion coefficient was systematically smaller. Individual cadherin trajectories were temporally heterogeneous, exhibiting alternating periods of fast and slow diffusion; the periods of slow diffusion (D slow z 0.1 mm 2 /s) were more prevalent at lower Ca 2þ concentration. These observations suggested that more flexible cadherin ectodomains at lower Ca 2þ concentration alternated between upright and lying-down conformations, where the latter interacted with more lipid molecules and experienced greater viscous drag.

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Section: Methodsmentioning

confidence: 99%

“…The soluble, expressed hexahistidinetagged ectodomains were first purified by affinity chromatography on an Affi-Gel column (Bio-Rad, Hercules, CA), as described in Sivasankar et al (28). Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the purity of the final protein.…”

Section: Methodsmentioning

confidence: 99%

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

Cai

Shashikanth

Leckband

et al. 2016

Biophysical Journal

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“…Sivasankar and colleagues, using a surface force apparatus, demonstrated that the strongest adhesive interaction between cadherins on two surfaces occurred at a minimum distance of about 25 nm (Sivasankar et al 1999;Leckband and Sivasankar 2000;Sivasankar et al 2001). Interestingly, this is the approximate length of a cadherin extracellular domain if all the repeats would be in a straight line, one after the other.…”

Section: Further Functional Studies Of Ec Domains In Adhesionmentioning

confidence: 99%

Cadherin Adhesion: Mechanisms and Molecular Interactions

Perez

Nelson

2004

Handbook of Experimental Pharmacology

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Cadherins constitute a superfamily of cell-cell adhesion molecules expressed in many different cell types that are required for proper cellular function and maintenance of tissue architecture. Classical cadherins are the best understood class of cadherins. They are single membrane spanning proteins with a divergent extracellular domain of five repeats and a conserved cytoplasmic domain. Binding between cadherin extracellular domains is weak, but strong cell-cell adhesion develops during lateral clustering of cadherins by proteins that link the cadherin cytoplasmic domain to the actin cytoskeleton. Understanding how different regions of cadherins regulate cellcell adhesion has been a major focus of study. Here, we examine evidence of the structure and function of the extracellular domain of classical cadherins in regard to the control of recognition and adhesive contacts between cadherins on opposing cell surfaces. Early experiments that focused on understanding the homotypic, Ca ++ -dependent characteristics of cadherin adhesion are discussed, and data supporting the widely accepted cis-and trans-dimer models of cadherins are analyzed.

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“…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). 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).…”

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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Direct molecular force measurements of multiple adhesive interactions between cadherin ectodomains

Cited by 160 publications

References 35 publications

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

Cadherin Adhesion: Mechanisms and Molecular Interactions

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

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