Analysis of the Function of Spire in Actin Assembly and Its Synergy with Formin and Profilin

Abstract

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.

Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation

“…In Drosophila oocytes, Spire and Cappuccino (the fly formin-2) are required for establishing oocyte polarity by organizing an actin network, although the precise function of the actin network differs from that present in mouse oocytes [Dahlgaard et al, 2007;Quinlan et al, 2007]. In vitro biochemical studies have also established the interactions of these two types of actin nucleators [Bosch et al, 2007;Quinlan et al, 2007], but it remains to be determined how formin-2 and Spire synergize in vivo to nucleate actin filaments.…”

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

“…The N-terminal domain of Spire (SpireNT, residues 1-520 in Drosophila melanogaster Spire; see Fig. S1) has the potential to form a string of four actin monomers through the interaction with four WH2 repeats (13,19,20). WH2 motifs are known to be intrinsically disordered, adopting an α-helical structure only upon binding to actin (23).…”

“…Spire contains four consecutive WH2 domains and is the most important representative member of the group. The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (17) and formins (18), whereas several different mechanisms have been proposed for Spire (19)(20)(21)(22), Leiomodin (15), JMY (6), and the APC protein (16). The N-terminal domain of Spire (SpireNT, residues 1-520 in Drosophila melanogaster Spire; see Fig.…”

“…Additionally, the L3 linker between domains C and D was identified as an actin monomer binding element with weak nucleating activity (6). In an actin assembly assay, the ABCD nucleating region of Spire nucleates actin at low Spire-actin ratios and sequesters actin at high Spire-actin ratios (7,13,19,20,27). It was concluded that the complexes of Spire with actin are formed in efficient and cooperative association reactions, are stable, and adopt the shape of one strand of the long-pitch helix of the actin filament, thus making a filament template (13,19,21,28).…”

“…In an actin assembly assay, the ABCD nucleating region of Spire nucleates actin at low Spire-actin ratios and sequesters actin at high Spire-actin ratios (7,13,19,20,27). It was concluded that the complexes of Spire with actin are formed in efficient and cooperative association reactions, are stable, and adopt the shape of one strand of the long-pitch helix of the actin filament, thus making a filament template (13,19,21,28). However, recent high-resolution crystallographic studies of a number of Spire-actin constructs composed of one to four WH2 domains including the linkers did not support this view and showed arrangements that significantly differed from the structure of actin filaments (20).…”