The multivalent carbohydrate-carbohydrate interaction between membrane anchored epitopes derived from the marine sponge Microciona prolifera (M. 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 ion concentration and loading rate. A deterministic model was used to estimate the number of participating bonds in the contact zone.

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.

Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.embrane fusion plays a pivotal role in many fundamental biological processes, comprising viral infection, cell-cell fusion during fertilization, tissue formation, and intracellular transport during exo-and endocytosis (1). Among the best-studied biological examples is the Ca 2+ -regulated fusion of synaptic vesicles with the presynaptic plasma membrane in neurons and chromaffin cells (2, 3). Fusion in these cells is catalyzed by concerted action of SNARE proteins through formation of a tetrameric coiled-coil complex that releases a sufficient amount of free energy to lower the barriers for membrane merging. One major energy barrier is associated with the strong repulsive hydration forces when two smooth bilayers approach each other. Other energy-costly contributions originate from lipid splaying as the initiation of stalk formation, expansion of the stalk structure driven by the strong curvature associated with membrane destabilization, hemifusion diaphragm expansion, and, finally, pore formation (4-8). The free energy associated with complex formation and folding of SNAREs was reported to be ∼35 k B T, close to the energy required to create the highly curved transition structures (40-50 k B T) (9, 10). Albeit a part of this free energy might be dissipated as heat, this coincidence of matching energy is also supported by experimental studies showing that only very few SNARE complexes are sufficient to induce fusion in artificial systems (5,6,(11)(12)(13). Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) m...

Weak noncovalent intermolecular interactions play a pivotal role in many biological processes such as cell adhesion or immunology, where the overall binding strength is controlled through bond association and dissociation dynamics as well as the cooperative action of many parallel bonds. Among the various molecules participating in weak bonds, carbohydrate-carbohydrate interactions are probably the most ancient ones allowing individual cells to reversibly enter the multicellular state and to tell apart self and nonself cells. Here, we scrutinized the kinetics and thermodynamics of small homomeric Lewis X-Lewis X ensembles formed in the contact zone of a membrane-coated colloidal probe and a solid supported membrane ensuring minimal nonspecific background interactions. We used an atomic force microscope to measure force distance curves at Piconewton resolution, which allowed us to measure the force due to unbinding of the colloidal probe and the planar membrane as a function of contact time. Applying a contact model, we could estimate the free binding energy of the formed adhesion cluster as a function of dwell time and thereby determine the precise size of the contact zone, the number of participating bonds, and the intrinsic rates of association and dissociation in the presence of calcium ions. The unbinding energy per bond was found to be on the order of 1 kBT. Approximately 30 bonds were opened simultaneously at an off-rate of koff = 7 ± 0.2 s(-1).