In vitro analysis of the functional differences between proteins of the ADF/cofilin family

Abstract

The actin depolymerizing factor (ADF)/cofilins (AC) are an essential group of actin filament depolymerizing and severing proteins that regulate actin filament turnover in vivo. In metazoans, two or more members are often expressed in the same cell. Each member has qualitatively similar interactions with actin, but differences have been reported in the regulation of their expression that suggests they may have different functions. We compared the interaction of chick ADF and chick cofilin with chick actin. The critical concentration of ADF-actin (6 μM) is much higher than that of cofilin-actin (0.8 μM) and actin alone (0.13 μM). The apparent dissociation constant of MgATP-actin and ADF measured by non-denaturing polyacrylamide gel electrophoresis is 1 μM. 10-fold lower than for cofilin. Cofilin promotes filament assembly whereas ADF does not. These data suggest that ADF is likely to be a sequestering protein for ATP-G-actin in vivo. Long filaments predominate at steady state in samples containing cofilin, whereas short filaments predominate in samples containing ADF, demonstrating that ADF severs and/or depolymerizes filaments to a greater extent. ADF severs and depolymerizes filaments from the pointed end faster than cofilin and ADF activity is pH-dependent. Severing is favored at pH 7.8 whereas enhancement of off-rate is favored at pH 6.8. ACs from other species behaved either like ADF or cofilin based on their interactions with G-actin and F-actin. The chick ADF-like group includes Xenopus XAC. and starfish depactin. The chick cofilin-like group includes Arabidopsis ADF 1, Caenorhabditis UNC-60B. Drosophila twinstar. Dictyostelium cofilin. and Acanthamoeba actophorin. Therefore, ADF and cofilin differ quantitatively in their interaction with actin. When actin monomer concentration is between the critical concentration of ADF-actin and cofilin-actin, ADF will depolymerize filaments whereas cofilin will promote filament assembly. These findings explain the different effects observed when ADF and cofilin are overexpressed in cells.