Characterisation of cellular adhesion reinforcement by multiple bond force spectroscopy in alveolar epithelial cells: Cell adhesion by multibond force spectroscopy

Background : Integrin-mediated adhesion is a key process by which cells physically connect their environment, express sensitivity and adaptation through mechanotransduction. A critical step of cell adhesion is the formation of the first bonds which individually generate weak contacts (∼tens pN) but can sustain thousand times higher forces (∼tens nN) when associated. Results : We propose an experimental validation by multiple bond force spectroscopy (MFS) of a stochastic model predicting adhesion reinforcement permitted by non cooperative, multiple bonds on which force is homogeneously distributed (called parallel bond configuration). To do so, spherical probes (diameter: 6.6 μm) specifically coated by RGD-peptide to bind integrins, are used to statically indent and homogenously stretch the multiple bonds created for short contact times (2s) between the bead and the surface of epithelial cells (A549). Using different separation speeds ( v = 2, 5, 10 μm/s) and measuring cellular Young's modulus as well as the local stiffness preceding local rupture events, we obtain cell-by-cell the effective loading rates both at the global cell level and at the local level of individual constitutive bonds. Local rupture forces are in the range: f* = 60 − 115pN while global rupture (detachment) forces reach F* = 0.8 − 1.7 nN. Global and local rupture forces both exhibit linear dependencies with the effective loading rate, the slopes of these two linear relationships providing an estimate of the number of independent integrin bonds constituting the tested multiple bond structure (∼12). Conclusions : The MFS method enables to validate the reinforcement of integrin-mediated adhesion induced by the multiple bond configuration in which force is homogeneously distributed amongst parallel bonds. Local rupture events observed in the course of a spectroscopy maneuver (MFS) lead to rupture force values considered in the literature as single-integrin bonds. Significance : Adhesion reinforcement permitted by the parallel multiple bond association is particularly challenging to verify for two reasons: first, it is difficult to control precisely the direction of forces experimentally, and second, because both global and local bond rupture forces depend on the effective loading rate applied to the bond. Here we propose an integrin-specific MFS method capable of detecting bond number and characterizing bond configuration and its impact on adhesion strength. This article is protected by copyright. All rights reserved