Elongated membrane tethers, individually anchored by high affinity α4ß1/VCAM-1 complexes, are the quantal units of monocyte arrests.

The α4ß1 integrin facilitates both monocyte rolling and adhesion to the vascular endothelium and is physiologically activated by monocyte chemoattractant protein (MCP-1). The current study investigated the initial events in the adhesion of THP-1 cells to immobilized Vascular Cell Adhesion Molecule 1 (VCAM-1). Using AFM force measurements, cell adhesion was shown to be mediated by two populations of α4ß1/VCAM-1 complexes. A low affinity form of α4ß1 was anchored to the elastic elements of the cytoskeleton, while a higher affinity conformer was coupled to the viscous elements of the cell membrane. Within 100 ms of contact, THP-1 cells, stimulated by co-immobilized MCP-1, exhibited a tremendous increase in adhesion to VCAM-1. Enhanced cell adhesion was accompanied by a local decoupling of the cell membrane from the cytoskeleton and the formation of long membrane tethers. The tethers were individually anchored by multiple α4ß1/VCAM-1 complexes that prolonged the extension of the viscous tethers. In vivo, the formation of these membrane tethers may provide the quantal structural units for the arrest of rolling monocytes within the blood vessels.

Force-clamp measurements of receptor-ligand interactions.

Protein-protein interactions are the basis of both biochemical and biophysical signaling of living cells. In many cases, the receptor is present on the cell surface while the ligand is in solution or linked to another support (extracellular matrix or another cell). In the case of cellular adhesion, forces are continuously applied to receptor-ligand complexes and, as a consequence, the dissociation kinetics of the bonds may change. It is, thus, relevant to study the kinetics of protein-protein interactions in response to applied forces, as this is the most physiologically relevant situation. The atomic force microscope (AFM) was one of the first nanotools to be applied to this end. However, new approaches need to be developed to better understand the complex energy landscape of molecular interactions under applied stress. In this chapter, we described the use of the AFM to carry out force-clamp measurements on receptor-ligand bonds. Force-clamp measurements on bonds consist of applying a constant and controlled force to a receptor-ligand bond and measure the resulting dissociation lifetime. The described methods include the required materials, functionalization of tips and substrates, force-clamping measurements, and processing and interpretation of the results. An illustrative example is given with the well-studied streptavidin-biotin complex.

Temperature modulation of integrin-mediated cell adhesion.

In response to external stimuli, cells modulate their adhesive state by regulating the number and intrinsic affinity of receptor/ligand bonds. A number of studies have shown that cell adhesion is dramatically reduced at room or lower temperatures as compared with physiological temperature. However, the underlying mechanism that modulates adhesion is still unclear. Here, we investigated the adhesion of the monocytic cell line THP-1 to a surface coated with intercellular adhesion molecule-1 (ICAM-1) as a function of temperature. THP-1 cells express the integrin lymphocyte function-associated antigen-1 (LFA-1), a receptor for ICAM-1. Direct force measurements of cell adhesion and cell elasticity were carried out by atomic force microscopy. Force measurements revealed an increase of the work of de-adhesion with temperature that was coupled to a gradual decrease in cellular stiffness. Of interest, single-molecule measurements revealed that the rupture force of the LFA-1/ICAM-1 complex decreased with temperature. A detailed analysis of the force curves indicated that temperature-modulated cell adhesion was mainly due to the enhanced ability of cells to deform and to form a greater number of longer membrane tethers at physiological temperatures. Together, these results emphasize the importance of cell mechanics and membrane-cytoskeleton interaction on the modulation of cell adhesion.

A streptavidin variant with slower biotin dissociation and increased mechanostability.

Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.