Laboratoire Denzymologie Et Of Biochimie Structurales

Gif-sur-Yvette, France

Laboratoire Denzymologie Et Of Biochimie Structurales

Gif-sur-Yvette, France

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Derivery E.,Laboratoire Denzymologie Et Of Biochimie Structurales | Gautreau A.,Laboratoire Denzymologie Et Of Biochimie Structurales
Communicative and Integrative Biology | Year: 2010

WASH is the Arp2/3 activating protein that is localized at the surface of endosomes, where it induces the formation of branched actin networks. This activity of WASH favors, in collaboration with dynamin, the fission of transport intermediates from endosomes, and hence regulates endosomal trafficking of several cargos. We have purified a novel stable multiprotein complex containing WASH, the WASH complex, and we examine here the evolutionary conservation of its seven subunits across diverse eukaryotic phyla. This analysis supports the idea that the invention of the WASH complex has involved the incorporation of an independent complex, the CapZ α/β heterodimer, forming the so-called Capping Protein (CP), as illustrated by the yeasts S. cerevisiae and S. pombe, which possess the CP heterodimer but no other subunits of the WASH complex. The alignements of the orthologous genes that we have generated give a view on the conservation of the different subunits and on their organization into domains. Moreover, we propose here a unique nomenclature for the different subunits to prevent future confusions in the field. © 2010 Landes Bioscience.


Derivery E.,Laboratoire Denzymologie Et Of Biochimie Structurales | Gautreau A.,Laboratoire Denzymologie Et Of Biochimie Structurales
Methods in Enzymology | Year: 2010

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. © 2010 Elsevier Inc.


PubMed | Laboratoire Denzymologie Et Of Biochimie Structurales
Type: | Journal: Methods in enzymology | Year: 2010

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.

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