Atomic force microscopy study of the integrin alpha 5beta1 and fibronectin interaction
Date of Award
Doctor of Philosophy (Ph.D.)
Physiology and Biophysics
First Committee Member
Vincent T. Moy, Committee Chair
The interaction of alpha5beta1 integrin and fibronectin (FN) plays a crucial role in the adhesion between cells and extracellular matrix. I have carried out atomic force microscopy (AFM) measurements to determine the dynamic response of the alpha5 beta1/FN complex to a pulling force.The dynamic rupture forces of individual alpha5beta 1/FN complexes in the presence of Mn2+ or Mg2+ revealed two loading regimes. High concentrations of Ca2+ suppressed the rupture forces of the complex and revealed only one loading regime. Force measurements on a FN RGD deleted-mutant revealed that the rupture forces were suppressed at both loading regimes. Mutations to the synergy site of FN lowered the rupture forces only at the slow loading regime. Activation of alpha5beta1/FN interactions by TS2/16 antibody elevated the rupture forces in the slow loading regime only. Interestingly, in the presence Mn2+ the rupture force of alpha5beta 1 and the synergy mutant complex was increased to the level of the wild type interaction after adding TS2/16.I demonstrated that the RGD sequence is essential for the single molecular interaction between alpha5beta1 and FN. At the high affinity state, the synergy site is not absolutely required for binding. My results supported a model in which the synergy site is not directly involved in binding, but may have an important role in stabilizing the interaction. Using a two-barrier Bell model, the fast and slow force loading regimes in the dynamic force spectra characterize the inner and outer activation barriers of the alpha5beta1/FN complex, respectively. The interaction between the RGD sequence and a bound divalent ion contributes to both the inner and outer activation barriers of the alpha5beta 1/FN complex. The synergy site contributes only to the outer barrier. The inner barrier is seen at high forces. The outer activation barrier operates at lower and physiologically relevant forces. My research demonstrates that both the RGD sequence and the synergy site are important for the adhesion between alpha5beta1 and FN. In addition, the outer barrier determines the fast dissociation of the alpha5beta 1/FN complex in response to the pulling force, which may be a biologically important feature during cell migration.
Li, Feiya, "Atomic force microscopy study of the integrin alpha 5beta1 and fibronectin interaction" (2004). Dissertations from ProQuest. 2119.