Actin Assembly on Biomimetic Lipid-coated and Functionalized Bead Surfaces

Abstract

Dynamic assembly of actin filaments drives cell protrusion and other aspects of cell motility. Reconstitution of actin assembly from purified components is a powerful approach for understanding the molecular mechanisms that underlie these processes. Purified preparations of actin, actin-binding proteins, and regulators of actin-binding proteins enable a broad spectrum of reconstitution experiments. Much of this actin filament assembly occurs at the plasma membrane, with exquisite temporal and spatial regulation. We developed a biomimetic reconstitution system using purified lipids or glutathione on the surface of functionalized beads as models for actin assembly at the plasma membrane. The system incorporates two critical pathways for regulation of actin assembly: nucleation of actin polymerization by Arp2/3 complex and activation of heterodimeric actin capping protein (CP). Arp2/3 complex and CP are both essentially inactive in the basal resting state of the cytoplasm. The system incorporates membrane-bound activators for Arp2/3 complex, such as WASP and WAVE family proteins, and for CP, such as CARMILs and other CPI-motif proteins. Membrane-bound components are coupled to beads either through lipids, which allow lateral mobility, or through glutathione groups attached to the bead surface via flexible linkers. Fluorescence microscopy of labeled components, combined with quantitative image analysis, is used to assess the combined effects of the regulators and thereby test molecular mechanisms for regulation of actin assembly. Here, we describe protocols for preparing functionalized beads, coupling regulators to the bead surface, performing actin assembly reactions, and imaging and quantifying the resulting actin networks.