In vitro actin assembly assays and purification from Acanthamoeba.

Abstract

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility and endocytosis. Actin polymerization is tightly regulated by various cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. The pyrene actin assembly assay is a powerful tool for determining the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleators or capping factors. In addition, fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how these proteins influence actin filament superstructure.