UCLA

The actin cytoskeleton is essential for cellular functions such as cell motility, division, and establishment and maintenance of cell shape. Just as important as the actin cytoskeleton are the proteins that stimulate filament assembly called nucleators. There are three classes of nucleators: the Arp2/3 complex, the formins, and the recently discovered Wasp Homology-2 (WH2)-family of nucleators. All three nucleate different types of actin structures and employ unique nucleating mechanisms to do so. Of the three classes of nucleators, we know the least about the WH2-nucleators. What we do know is that WH2-nucleators use a mechanism involving tandem WH2 domains to initiate actin filament assembly. Although the WH2-nucleators share a common domain, the current literature suggests they nucleate using different mechanisms. Previous studies suggest that Spir, a WH2-nucleator unique to metazoans and essential for polarity in developing oocytes and embryos, nucleates filaments by forming a linear nucleus or protofilament. Here we use a biochemical approach to further investigate the mechanism of nucleation and to identify how Spir differs from other non-nucleating, WH2-containing proteins such as N-Wasp. We investigated the biochemical features of Spir and N-Wasp WH2 domains interacting with actin under both steady state and kinetic conditions. Despite their short and similar sequences, all WH2 domains show distinct effects on the multiple phases of actin assembly. We found that the third WH2 domain in Spir, WH2C, is unique and specialized for nucleation in its sequence and interaction with both monomeric and filamentous actin. Work with tandem nucleating constructs revealed that the order of the WH2 domains in Spir influence nucleation activity and the ability to cooperatively bind actin.