Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins.

A minimal level of viscoelasticity in the cytoskeleton is an essential prerequisite of cellular motility. To determine the influence of the F-actin crosslinking proteins alpha-actinin and actin-binding protein (ABP)120 gelation factor from Dictyostelium discoideum on the properties of actin gels we used a torsion pendulum to measure directly viscoelastic changes of the filamentous networks. Using the capping proteins severin and DS151 to control actin filament length, both crosslinkers were found to increase the elasticity and the viscosity of F-actin solutions. In the case of alpha-actinin, this activity was completely blocked by micromolar concentrations of Ca2+. The inhibitory functions of the two EF hands of alpha-actinin were further investigated by introducing point mutations into either one or both of the Ca(2+)-binding regions. Mutations in the Ca(2+)-coordinating amino acid residues in the first or in both EF hands left the dynamic storage and loss moduli of the F-actin solution unaltered, independent of the Ca2+ concentration. However, alpha-actinin mutated in the second EF hand increased the viscoelasticity of actin gels like the wild-type protein in the absence of Fa2+. The ABP120 gelation factor exhibited only negligible differences to alpha-actinin in viscometry measurements, whereas its impact on the ratio G"/G' (the ratio of energy lost compared to elastically stored during a deformation) of F-actin solutions was clearly smaller than that of alpha-actinin. We conclude from these data that: (i) a torsion pendulum is an excellent tool to determine small changes of activity in normal and mutated actin-binding proteins, (ii) the first EF hand of alpha-actinin is crucial for its crosslinking function, and (iii) the viscoelastic properties of F-actin gels crosslinked by either alpha-actinin or the ABP120 gelation factor are different.