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 properti es of actin gels we used a torsion pendulum to measure directly viscoe lastic 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 so lutions. In the case of alpha-actinin, this activity was completely bl ocked by micromolar concentrations of Ca2+, The inhibitory functions o f the two EF hands of alpha-actinin were further investigated by intro ducing point mutations into either one or both of the Ca2+-binding reg ions, Mutations in the Ca2+-coordinating amino acid residues in the fi rst or in both EF hands left the dynamic storage and loss moduli of th e F-actin solution unaltered, independent of the Ca2+ concentration, H owever, alpha-actinin mutated in the second EF hand increased the visc oelasticity of actin gels like the wild-type protein in the absence of Ca2+, The ABP120 gelation factor exhibited only negligible difference s to alpha-actinin in viscometry measurements, whereas its impact on t he ratio G ''/G' (the ratio of energy lost compared to elastically sto red during a deformation) of F-actin solutions was clearly smaller tha n that of alpha-actinin. We conclude from these data that: (i) a torsi on pendulum is an excellent tool to determine small changes of activit y 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 alph a-actinin or the ABP120 gelation factor are different. (C) 1996 Academ ic Press, Inc.