Covalent and Density-Controlled Surface Immobilization of E-Cadherin for Adhesion Force Spectroscopy

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.

N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells

“…2), albeit at levels much reduced when compared to RGD-conjugated gels (HAVDI attachment ~10% of RGD attachment). This is consistent with previous reports showing that the first two extracellular domains of N-Cadherin (in which the HAVDI sequence resides) allow for weak adhesive interactions when compared to the full ectodomain 41 . Next, we examined 1 mM: 1 mM peptide combinations of HAVDI and RGD across a physiologic range of substrate stiffness, and noted no significant difference in spread area between HAVDI/RGD and scram/RGD groups (Fig.…”

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness

“…While classic trans cadherin bonds are mediated by homophilic strand swapping of an N-terminal tryptophan residue into a hydrophobic pocket of its binding partner 13 , we previously demonstrated that the trans tip-link bond has a far more extensive, amphiphilic "handshake" interface involving ~30 amino acids in the first two extracellular (EC) domains of each protein 8 . Although the dissociation constant of a single tip-link bond is approximately the same as for classic cadherins 8,[14][15][16][17][18] , little is known about how the tip-link bond responds to force.…”

“…1f). Compared with classic cadherins (mean lifetime ≈ 0.75 s, fβ ≈ 5.5 pN) [14][15][16][17][18] , the single tip-link bond is significantly more resistant to mechanical force (Fig. 1g).…”

Cell adhesion and urothelial bladder cancer: the role of cadherin switching and related phenomena

“…Traditionally, cell-cell adhesion is described as being achieved by the symmetric interactions of the first extracellular domains (EC1) of cadherins on neighbouring cells (trans-interaction) [63,66]; cadherins on the same cell also interact with each other (cis-interaction) through the EC1 domain of one and the EC2 domain of the other [63,66,67]. More recently, it has been described that optimal cell-cell adhesion (50-70 pN) is achieved by all five EC domains of E-cadherin, and with a cellcell separation of 5-11 nm [64] ( figure 1). E-, P-and N-cadherin were the first cadherins identified, and can all mediate cell-cell adhesion in this fashion [62,68]: -E-cadherin (cadherin-1, 120 kDa): the main mediator of cell -cell adhesion in epithelial tissues and expressed by most normal epithelial cells [19,60,61,[68][69][70].…”

“…Traditionally, cadherins on neighbouring cells adhere via EC1 domains, although more recent research suggests that all five EC domains are required for optimal adhesion [64]. Figure 2.…”