Regulation of cell adhesion strength by peripheral focal adhesion distribution

Abstract

Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength.

Figure 1

Schematic diagram of cells adhered to micropatterned islands that delineate cell adhesive area and cell spreading area.

Figure 2

Immunostained image of a (a) spread cell (color online: blue, nucleus; red, f-actin; and green, vinculin). (b) Same cell thresholded for peak intensities of vinculin staining (bars = 10 μm).

Figure 3

Immunostaining indicates fibronectin adsorbed only to micropatterned islands: (a) 6-μm-diameter circular islands; (b) 10-μm-outer, 8-μm-inner-diameter annulus islands; (c) 10-μm-diameter circular islands; (d) 25-μm-outer, 23-μm-inner-diameter annulus islands; and (e) 25-μm-diameter circular islands (bars = 50 μm).

Figure 4

(a–c) Solid circular and (d–f) annular islands were coated with (a and d) fibronectin to regulate cell spreading and focal adhesion assembly. (b and e) Adherent cells were immunostained to identify adhesive structures (color online: blue, nucleus; red, f-actin; and green, vinculin). (c and f) Images were thresholded for the peak intensities of vinculin staining (bars = 10 μm).

Figure 5

Mean adhesion strength (τ₅₀) at steady state for cells patterned on micropatterned domains in (a) regime 1 up to 78 μm² adhesive area and (b) regime 2 from 78 μm² to 490 μm² adhesive area (asterisk indicates significant difference P < 0.001).

Figure 6

Cell-spreading area and cell-adhesive area each regulates steady-state adhesion strength. Data are plotted separately for circular and annular islands of two corresponding adhesive areas. Exponential curves describe the relationships between adhesion strength and spreading for the different focal adhesion distribution conditions. Symbols represent mean values, but the curves were fit to all data points.

Figure 7

Experimental cell adhesion strength-spreading relationship for peripherally distributed focal adhesions agrees well with theoretical predictions of the adhesive patch model. Data are plotted separately for circular and annular islands. Exponential curves describe the relationships between adhesion strength and spreading for the different focal-adhesion distribution conditions. Symbols represent mean values but the curves were fit to all data points.