Cellular attachment to the extracellular matrix (ECM) via integrin cell surface receptors is essential for signaling of the most basic of biological function such as apoptosis, differentiation and motility (Hynes, 1992).

Divalent cations play a critical role in the α5β1-fibronectin interaction as evidenced by (1) regulation of the affinity of interaction by cations [e.g., Ca²⁺ down regulates and Mg²⁺ or Mn²⁺ up regulates α5β1 binding affinity (Gailit, 1988; Mould, 1995)] and (2) loss of molecular interaction between α5β1 and fibronectin upon chelation using EDTA (Mould, 1995; Li, 2003).

The primary goal of the present study was to investigate the mechanisms underlying the cation-induced changes in α5β1-fibronectin interaction. We used atomic force microscopy (AFM) to directly examine the α5β1-fibronectin interaction in the presence of affinity regulating cations (i.e. Ca ²⁺, Mg²⁺, Mn²⁺, CaMg or CaMn) and at load rates commensurate with known cellular motility speeds.

The rupture force was linearly proportional to load rate and plotted data for this relationship corresponded to a single line resulting from limited bond separation resistance for down regulated α5β1 (Ca²⁺ , CaMg); or two lines (the second showing rapidly increasing rupture force) resulting from up regulated α5β1 (CaMn, Mg²⁺, Mn²⁺). A sole ‘outer barrier’ dominated bond separation resistance for down regulation and low load rates (<10,000 pN/s) while up regulation produced the additional, second barrier (inner barrier), which dominated resistance at high load rates (>10,000 pN/s).

No significant difference in bond rupture force (P = 0.68) existed at low load rates between down and up regulated α5β1, since each encountered the same resistance (outer barrier). However, only the up regulated form of α5β1 encountered the additional energy barrier at high load rates, resulting in sharply increasing forces.

Although both Mg²⁺ and Mn²⁺ up regulated α5β1, the addition of Ca²⁺ down regulated α5β1, resulting in the elimination of the second (inner barrier) only for Mg²⁺; it was unable to do so for Mn²⁺.

Overall, these results support the premise that a cation related mechanism is responsible for affinity regulation of α5β1 leading to different bond separation resistance at low and high load rates.