In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a ping-pong mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase-independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle. Author Summary Mammalian reproduction requires successful meiosis, which consists of two strongly asymmetric cell divisions. In meiosis I, movement of the spindle (the subcellular structure that segregates chromosomes during division) toward the oocyte cortex (the outer layer of the egg) is essential for fertility. This process requires that actin filaments assemble in a dynamic mesh, driven by three actin binding proteins, profilin, formin 2, and Spire. To date the molecular mechanisms by which these three proteins cooperate are not known. We now explore this in vitro by a combination of bulk solution and single actin filament assembly assays in the presence of profilin, Spire, and formin 2. Individually, Spire binds to actin filament ends to block their growth, and by itself, formin 2 associates poorly with filament ends, promoting fast processive assembly from the profilin-actin complex. However, when present together, Spire and formin 2 interact with one another (the formin 2 C-terminal binds to the N terminal Spire KIND domain), forming transient complexes at filament ends from which each binds alternately to the filament ends to regulate actin assembly by a ping-pong mechanism. Our in vitro observations are validated by injection studies in mouse oocytes. In oocytes, the additional interaction of Spire and formin 2 with Rab11a-myosin Vb vesicles couples high actin dynamics to vesicle traffic.
