Rapid stiffening of integrin receptor-actin linkages in endothelial cells stimulated with thrombin: A magnetic bead microrheology study

By using magnetic bead microrheology we study the effect of inflammatory ag ents and toxins on the viscoelastic moduli of endothelial cell plasma membr anes in real time. Viscoelastic response curves were acquired by applying s hort force pulses of similar to 500 pN to fibronectin-coated magnetic beads attached to the surface membrane of endothelial cells. Upon addition of th rombin, a rapid stiffening of the membrane was observed within 5 s, followe d by recovery of the initial deformability within 2 min. By using specific inhibitors, two known pathways by which thrombin induces actin reorganizati on in endothelial cells, namely activation of Ca2+-calmodulin-dependent myo sin light chain kinase and stimulation of Rho/Rhokinase, were excluded as p ossible causes of the stiffening effect. Interestingly, the cytotoxic necro tizing factor of Escherichia coli, a toxin which, in addition to Rho, activ ates the GTPases Rac and CDC42Hs, also induced a dramatic stiffening effect , suggesting that the stiffening may be mediated through a Rac- or Cdc42Hs- dependent pathway. This work demonstrates that magnetic bead microrheometry is not only a powerful tool to determine the absolute viscoelastic moduli of the composite cell plasma membrane, but also a valuable tool to study in real time the effect of drugs or toxins on the viscoelastic parameters of the plasma membrane.