Temporal effect of functional blocking of β₁ integrin on cell adhesion strength under serum depletion

Cell adhesion is generally concomitant to the formation of focal adhesion. Although it is well-known that focal adhesion plays an important role in the functional regulations of anchorage dependent cells, previous experimental studies have not provided quantitative description of the relation between focal adhesion and biophysical responses of cells. Furthermore, there is lack of knowledge on the importance of the β₁ integrin subunit to the dynamic responses of cells during initial cell seeding. In this study, we attempt to bridge the quantitative connection between focal adhesion density and cell-substrate interactions and evaluate the influence on functional blocking of β₁ integrin on adhesion strength. Total internal reflection fluorescence microscopy (TIRFM), fluorescence microscopy, and phase contrast microscopy was employed to study the time-dependent evolvement of vinculin pattern, distribution of actin filament, and morphological change, respectively, during 4 h of culture for porcine esophageal fibroblasts (non-blocked and β₁-blocked) on a fibronectin-coated surface. Micropipet aspiration technique was used to study the change of mechanotransduction through the determination of adhesion force and strength. It is shown in our experimental results that spread area, adhesion force, and adhesion strength increases over time on the two types of cells. Throughout the culture period, the two key mechanotransduction parameters of non-blocked cells is higher than those of β₁-blocked cells. Interestingly, adhesion strength initially ascends, then begins to diminish at a critical time point, and finally resumes increasing linearly against the increase of focal adhesion density. This variation as mentioned above can be explained by peeling and fracture models based on the dissimilar vinculin pattern of cells after being cultured for different time periods. Moreover, the averaged focal adhesion strength and non-focal adhesion strength of β₁-blocked cells are significantly less than those of non-blocked of cells. The weaker adhesion strength on β₁-blocked cells is directly caused by lower focal and non-focal adhesion strength, as well as by smaller focal adhesion density.