Abstract
E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5–11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.
Highlights
E-cadherin is the best-studied member among the calciumdependent cell-cell adhesion molecules
Domain monomers at defined surface density. To this end we generated two different constructs fused to a SNAP-12 histidine residues (12His)-tag in order to make use of the benzylguanine thiol (BGT) self-assembly monolayer (SAM) method we have developed previously [23]
A furin-like protease removes the cadherin propeptide at the cell surface, which limits the production of the cadherin fusion proteins to eukaryotic cell lines, as the protease is absent in bacteria
Summary
E-cadherin is the best-studied member among the calciumdependent cell-cell adhesion molecules. Homophilic binding between E-cadherins from neighboring cells organizes cells into epithelia which is essential for morphogenetic processes during embryonic and organ development and for maintaining tissue integrity and homeostasis [1]. The adhesive function of E-cadherin is required in stem cell renewal [2,3]. Loss of the E-cadherin function correlates with tumorigenesis [4], embryonic lethality [5,6] and loss of pluripotency of embryonic stem cells [7]. Cadherin adhesion has been reported to participate in mechanosensing by binding to plakoglobin and recruiting keratin filaments to sites of tension at the cell membrane [9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
