The Wave complex is intrinsically inactive.
The Wave proteins activate the Arp2/3 complex at the leading edge of migrating cells. The resulting actin polymerization powers the projection of the plasma membrane in lamellipodia and membrane ruffles. The Wave proteins are always found associated with partner proteins. The canonical Wave complex is a stable complex containing five subunits. Even though it is well admitted that this complex plays an essential regulatory role on Wave function, the mechanisms by which Wave proteins are regulated within the complex are still elusive. Even the constitutive activity or inactivity of the complex is controversial. The major difficulty of these assays resides in the long and difficult purification of the Wave complex by a combination of several chromatography steps, which gives an overall low yield and increases the chance of Wave complex denaturation. Here we report a greatly simplified approach to purify the human Wave complex using a stable cell line expressing a tagged subunit and affinity chromatography. This protocol provided us with sufficient amount of pure Wave complex for functional assays. These assays unambiguously established that the Wave complex in its native conformation is intrinsically inactive, indicating that, like WASP proteins, Wave proteins have a masked C-terminal Arp2/3 binding site at resting state. As a consequence, the Wave complex has to be recruited and activated at the plasma membrane to project migration structures. Importantly, the approach we describe here for multiprotein complex purification is likely applicable to a wide range of human multiprotein complexes.
Derivery
Emmanuel
E
Institut Curie, Centre de Recherche, Laboratory of Cell Morphogenesis and Intracellular Signaling, 75248 Paris Cedex 05, France.
Lombard
Bérangère
B
Loew
Damarys
D
Gautreau
Alexis
A
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes; D011554:Pseudopodia / Q000378:metabolism; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000302:isolation & purification / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D046912:Multiprotein Complexes; D051300:Wiskott-Aldrich Syndrome Protein Family; D020744:rac GTP-Binding Proteins