Assaying WAVE and WASH complex constitutive activities toward the Arp2/3 complex

Abstract

The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPFs). Among these, WAVE proteins are required for lamellipodia and ruffle formation, whereas WASH proteins are required for the fission of endosomes. Both WASH and WAVE NPFs are embedded into multiprotein complexes that provide additional functions and regulations. Understanding how these complexes regulate the activity of their NPF starts with the determination of the constitutive activity of the complex. In this chapter, we describe how to efficiently purify the WAVE and WASH complexes from human stable cell lines. We also describe how to verify that these complexes are not aggregated, a prerequisite for activity assays. We then provide a protocol to measure their activity toward the Arp2/3 complex using the well-established pyrene actin assay. Finally, we show how our fast purification protocol can be modified to detect the endogenous activity of the WAVE complex, providing an easy readout for the level of WAVE activation in cells.