The effects of detergents on the polymerization properties of actin

Abstract

Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes.