In vitro reconstitution of cdc42-mediated actin assembly using purified components.

In the accompanying chapter, we describe an in vitro system that uses Xenopus egg extracts to study actin assembly induced by phosphatidylinositol (4,5)bisphosphate (PIP2) and Cdc42. Biochemical fractionation and candidate screening experiments conducted in the extract system have identified the Arp2/3 complex, the N-WASP-WIP (or N-WASP-CR16) complex, and the Cdc42-binding protein Toca-1 as important mediators of PIP2- and Cdc42-actin signaling. Toward our ultimate goal of reconstituting an in vitro system that recapitulates the signaling properties observed in vivo, we then developed a purified actin assembly assay system consisting of the regulatory components that we discovered from extracts. In these assays, the stereotypical sigmoidal kinetics of actin polymerization are monitored by pyrene-actin fluorescence in the presence of defined recombinant or purified proteins, enabling the detailed study of mechanism and protein function. In this chapter, we describe the preparation of the components used in these purified actin assembly reactions, as well as the assay conditions under which we monitor actin polymerization kinetics in vitro.

Biochemical properties and inhibitors of (N-)WASP.

The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho GTPase Cdc42 and a key component of signaling pathways that regulate the actin cytoskeleton. WASP is regulated by a number of ligands, and the mechanisms by which these act are beginning to be understood through detailed biochemical analyses. Here we describe the protocols we use to study WASP proteins, including the methods we use to purify signaling components and the assays we use to quantitatively characterize the biochemical and biophysical properties of WASP, its activation by Cdc42, and its inhibition by the small molecule wiskostatin. These methods have broad use within the WASP-related cytoskeletal-signaling pathway but are also applicable to investigations of other intramolecular and intermolecular interactions.

Actin-based motility assay.

Actin-based movement can be reconstituted by using microspheres functionalized with the enzymes N-WASP or ActA, which use the Arp2/3 complex and actin to catalyze the formation of a branched actin filament network that is maintained in rapid turnover by three proteins (capping protein, profilin, and ADF). The particles continuously initiate filament assembly at their surface and are propelled, mimicking bacteria or the leading edge of motile cells. This biomimetic assay offers advantages over approaches based on living cells and cell extracts, because the physical-chemical parameters are under control. The biomimetic motility assay offers the opportunity to test the function of proteins involved in signaling pathways or actin dynamics. It is a powerful tool to understand the physical mechanism of force production and has the potential to support high-throughput screens for drugs, inhibitors of motility, or therapeutic agents in metastatic states in which motility is impaired.