Gelsolin as a calcium‐regulated actin filament‐capping protein

Various concentrations of gelsolin (25–100 nm) were added to 2 µm polymerized actin. The concentrations of free calcium were adjusted to 0.05–1.5 µm by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nm) that exceed the concentration of filament ends (≈ 2 nm), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointe