Force-induced growth of adhesion domains is controlled by receptor mobility.

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.

Force-controlled equilibria of specific vesicle-substrate adhesion.

We have developed "vertical" magnetic tweezers capable of exerting controlled pico and subpico Newton forces. Using this apparatus, we apply a point-like force to a vesicle that is adhered by means of specific bonds between the vesicle-carrying oligosaccharide sialyl LewisX and the surface-grafted E-selectin. An exponential decrease of the bound vesicle area with the decay rate that is insensitive to the force and the composition of the system is observed. We measure an equilibrium under force that is associated with an increased binding in the center of the contact zone. We also show that the determination of the shape is potentially sufficient to determine the number of formed specific bonds.

Absolute interfacial distance measurements by dual-wavelength reflection interference contrast microscopy.

Dual-wavelength reflection interference contrast microscopy (DW-RICM) is established as a microinterferometric technique to measure absolute optical distances between transparent planar substrates and hard or soft surfaces such as colloidal beads or artificial and biological membranes, which hover over the substrate. In combination with a fast image processing algorithm the technique was applied to analyze the trajectories of colloidal beads sedimenting under gravity. As the beads approach the surface of the substrate, they slow down because of hydrodynamic coupling of the bead motion to the substrate. The effective surface friction coefficients were measured as a function of the absolute distance of the beads from the surface. The height dependence of the friction coefficient was found to be in quantitative agreement with previous theoretical predictions. Furthermore, we demonstrate that the DW-RICM technique allows the determination of the height of membranes above substrates and the amplitude and direction of height fluctuations. Without any further need to label the membrane the unambiguous reconstruction of the surface profile of soft surfaces is possible.

Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion.

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicle's contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.