Direct Measurement of Thermodynamic Parameters of Cofilin-Actin Filament Interactions at the Single Molecule Level

Abstract

ADF/cofilin proteins are widely distributed in various species, and modulate actin dynamics by severing actin filaments. EM and biochemical analyses show that cofilin binds to actin filaments with positive cooperativity, and increases the twist of actin filaments. The phosphorescence anisotropy analysis shows the binding of cofilin increases the torsional fluctuation of actin filaments, and phalloidin inhibits the increase in the fluctuation induced by cofilin binding. We directly examined the binding of fluorescence-labeled cofilin to actin filaments by a single molecule imaging technique. The on rate of cofilin binding to actin filaments was estimated at 0.07 μM−1s−1, and the off-rate 0.6 s−1 when the cofilin concentration was 30-100 nM. During long-lasting cofilin bindings (>0.4 s), additional cofilin bindings in the vicinity (<300nm) of the initial binding site were observed, and the on-rate of the additional bindings was increased to 0.16 μM−1s−1. By contrast, the off-rate of the additional cofilin binding was not affected by the long-lasting binding. The cooperative parameter was estimated at 2.3 (0.16/0.07). These results indicate that long-lasting cofilin binding enhances the additional binding of cofilin in the vicinity of the initial binding site, but it does not affect the dissociation of cofilin. At 5 μM phalloidin, the on-rate of binding of cofilin to actin filaments decreased from 0.07 to 0.026 μM−1s−1, and the on-rate of the additional binding in its vicinity decreased from 0.16 to 0.032 μM−1s−1, while the off-rate of the initial and additional cofilin bindings were not altered; i.e., both the cofilin binding and the cooperative parameter (0.032/0.026=1.2) were decreased by phalloidin. Above results support the view that binding of cofilin increases the torsional fluctuation of actin filaments and the increase is required for the cooperative binding of cofilin.