Covalent and density-controlled surface immobilization of E-cadherin for adhesion force spectroscopy.

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.

Impact of peptide clustering on unbinding forces in the context of fusion mimetics.

Coiled-coil zipping and unzipping is a pivotal process in SNARE-regulated membrane fusion. In this study we examine this process mediated by a minimal model for coiled-coil formation employing force spectroscopy in the context of membrane-coated surfaces and probes. The interaction forces of several hundred pN are surprisingly low considering the proposed amount of molecular bonds in the contact zone. However, by means of high-resolution imaging employing atomic force microscopy and studying the lateral mobility of lipids and peptides as a function of coiled-coil formation, we are able to supply a detailed view on processes occurring on the membrane surfaces during force measurements. The interaction forces determined here are not only dependent on the peptide concentration on the surface, but also on the regional organization of lateral peptide clusters found prior to coiled-coil formation.

Model system for cell adhesion mediated by weak carbohydrate-carbohydrate interactions.

The multivalent carbohydrate-carbohydrate interaction between membrane-anchored epitopes derived from the marine sponge Microciona prolifera has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane-coated surfaces was employed to mimic native cell-cell contacts.The dynamic strength of the homomeric self-association was measured as a function of calcium ions and loading rate. A deterministic model was used to estimate the basic energy landscape and number of participating bonds in the contact zone.

Colloidal probe microscopy of membrane-membrane interactions: from ligand-receptor recognition to fusion events.

A versatile model system to study membrane-membrane interactions in great detail is introduced. Based on colloidal probe microscopy with membrane covered spherical probes attached to tip-less cantilevers the interaction forces and adhesion energies are quantified down to single molecule resolution. Two opposing membranes equipped with ligands on one side and receptors on the other side were brought in contact at a defined load and pulled apart at constant velocity. Ni-NTA functionalized lipids served as receptors on the probe, while lipopeptides displaying short His-tags (CGGH(6) or CGWH(6)) were incorporated in the planar supporting membrane on a silicon substrate. The rather intricate force distance curves were scrutinized in terms of breakthrough events upon contact of the probe with the surface, the overall work of adhesion, maximum adhesion force, as well as formation frequency, lifetime, and force of membrane tethers suggesting that hemifusion of the two opposing bilayers takes place.

Elasticity mapping of pore-suspending native cell membranes.

The mechanics of cellular membranes are governed by a non-equilibrium composite framework consisting of the semiflexible filamentous cytoskeleton and extracellular matrix proteins linked to the lipid bilayer. While elasticity information of plasma membranes has mainly been obtained from whole cell analysis, techniques that allow addressing local mechanical properties of cell membranes are desirable to learn how their lipid and protein composition is reflected in the elastic behavior on local length scales. Introduced here is an approach based on basolateral membranes of polar epithelial Madin-Darby canine kidney (MDCK) II cells, prepared on a highly ordered porous substrate that allows elastic mapping on a submicrometer-length scale. A strong correlation between the density of actin filaments and the measured membrane elasticity is found. Spatially resolved indentation experiments carried out with atomic force and fluorescence microscope permit relation of the supramolecular structure to the elasticity of cellular membranes. It is shown that the elastic response of the pore spanning cell membranes is governed by local bending modules rather than lateral tension.