Derivery Emmanuel, Gautreau Alexis
Laboratoire d’Enzymologie et de Biochimie Structurales, Gif sur Yvette, France.
Methods Enzymol. 2010;484:677-95. doi: 10.1016/B978-0-12-381298-8.00033-2.
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.
当被成核促进因子（NPFs）激活时，Arp2/3复合物会生成分支状肌动蛋白网络。其中，波状蛋白（WAVE蛋白）是片状伪足和褶皱形成所必需的，而WASH蛋白是内体分裂所必需的。WASH和WAVE NPFs都嵌入到多蛋白复合物中，这些复合物提供了额外的功能和调控。了解这些复合物如何调节其NPF的活性，首先要确定复合物的组成活性。在本章中，我们描述了如何从人稳定细胞系中高效纯化WAVE和WASH复合物。我们还描述了如何验证这些复合物没有聚集，这是活性测定的前提条件。然后，我们提供了一个使用成熟的芘肌动蛋白测定法来测量它们对Arp2/3复合物活性的方案。最后，我们展示了如何修改我们的快速纯化方案以检测WAVE复合物的内源性活性，为细胞中WAVE激活水平提供一个简单的读数。
