Integrin-mediated cell adhesion to fibronectin: initial binding and strengthening responses

Cell adhesion to fibronectin involves integrin receptor-ligand binding and adhesion strengthening, which includes integrin clustering, interactions with cytoskeletal components to form focal adhesions, and spreading. Because of the complex nature of the strengthening process, quantitative analyses of cell adhesion have been limited to initial events. In this study, we applied micropatterning methods to control focal adhesion size and decouple focal adhesion assembly from gross changes in cell morphology, allowing for rigorous analysis of adhesion strength independently of cell spreading and redistribution of adhesive structures. Microcontact printing was used to pattern arrays of circular adhesive islands within a non-adhesive background. Fibroblasts adhered to fibronectin-coated islands and remained nearly spherical. The cell-substrate contact area was constrained to the micropatterned domain and immunofluorescence staining revealed robust assembly of adhesive structures containing components associated with conventional focal adhesions. Cell adhesion strength to fibronectin-coated islands was quantified using a spinning disk device. Adhesion strength exhibited significant time- and adhesive area-dependent increases. Comparison of experiments for similar contact areas at different time points showed a 9-fold increase in adhesion strength over time, independently of cell spreading. This work provides an experimental framework for the functional analysis of focal adhesion components in physiological and pathological conditions.