SummaryOocytes mature into eggs by extruding half of their chromosomes in a small cell termed the polar body. Asymmetric oocyte division is essential for fertility [1], but despite its importance, little is known about its mechanism. In mammals, the meiotic spindle initially forms close to the center of the oocyte. Thus, two steps are required for asymmetric meiotic division: first, asymmetric spindle positioning and second, polar body extrusion. Here, we identify Spire1 and Spire2 as new key factors in asymmetric division of mouse oocytes. Spire proteins are novel types of actin nucleators that drive nucleation of actin filaments with their four WH2 actin-binding domains [2–6]. We show that Spire1 and Spire2 first mediate asymmetric spindle positioning by assembling an actin network that serves as a substrate for spindle movement. Second, they drive polar body extrusion by promoting assembly of the cleavage furrow. Our data suggest that Spire1 and Spire2 cooperate with Formin-2 (Fmn2) to nucleate actin filaments in mouse oocytes and that both types of nucleators act as a functional unit. This study not only reveals how Spire1 and Spire2 drive two critical steps of asymmetric oocyte division, but it also uncovers the first physiological function of Spire-type actin nucleators in vertebrates.

Abstract:The diversity of cellular actin functions is attained by the activation of actin nucleator complexes, which initiate the polymerization of actin monomers into a helical double-stranded filament at defined subcellular compartments. Next to actin functions at the cell membrane, including different forms of membrane protrusions and invaginations, actin dynamics at intracellular membranes has recently become a research focus. Experiments addressing the vesicle-associated Spir WH2 domain containing actin nucleators have provided novel mechanistic insights into the function of actin dynamics at intracellular membranes. Spir proteins are targeted by a modified FYVE zinc finger motif toward endosomal and vesicle membranes, where they interact and cooperate with the distinct nucleators of the FMN subfamily of formins in the nucleation of actin filaments. The function of the Spir/ formin actin nucleator complex is closely related to the Rab11 small G protein, which is a key regulator of recycling and exocytic transport processes. Together with the actin motor protein and Rab11 effector myosin Vb, Spir/ formin nucleated actin filaments mediate actin-dependent vesicle transport processes. Drosophila and mouse genetic studies as well as cell biology experiments point toward an important role of the Spir/formin complex in oocyte maturation and in the structure and signaling of the nervous system.

The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/ formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.The dynamic arrangement and rearrangement of the actin cytoskeleton are mandatory for cells to fulfill an impressive spectrum of tasks such as cell migration, vesicular transport processes, reorganization of cellular substructures, or the regulation of morphological dynamics. The amazing diversity of these cellular actin functions is reflected in a rapidly increasing number of regulatory mechanisms directing the dynamic assembly and disassembly of actin filaments (1). A specific prerequisite to set up the distinct actin structures is the spatial and temporal regulation of filament initiation. Due to the relative instability of the actin dimer and monomeric G-actin-binding proteins, the initiation of actin polymerization from free monomers (nucleation) requires factors that help to overcome the kinetic barrier to nucleation (1).Three different classes of actin nucleation factors have been described so far that function in the nucleation of actin filaments, firstly the actin-related protein 2/3 (Arp2/3) complex, secondly nucleation factors of the formin superfamily, and thirdly nucleation factors containing from one to multiple WH2 domains (2). Spir proteins are the founding members of the latter class of actin nucleation factors, the Wiskott-Aldrich syndrome protein homology 2 (WH2) domain-containing nucleators (3, 4). Spir nucleation activity resides in a cluster of four actin-binding WH2 domains in the central region of the protein (3). Althou...

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.The founding member of the RAF family of protein serine/ threonine kinases was discovered as the oncogene of mouse sarcoma virus 3611 (35). In vertebrate species, three RAF genes (A-RAF, B-RAF, and C-RAF) have been identified (5,14). B-RAF-deficient mice die between embryonic day 11.5 (E11.5) and E12.5 due to vascular hemorrhaging caused by increased apoptosis of endothelial cells (50). These animals also suffer from neuronal cell death (46) and a range of other defects that arise as a consequence of a significant disruption to ERK activation in these cells (48). Our earlier work further established that B-RAF is the major MEK activator in vivo and that C-RAF is required for normal B-RAF function (48). Targeted disruption of A-RAF or C-RAF genes demonstrated that their functions are not fully redundant with B-RAF, since null mutations for each gene resulted in distinct phenotypes (18,24,30,(48)(49)(50).Knock-in experiments support an important role of C-RAF in apoptosis suppression (6,18,53). The presence of multiple interaction partners of RAF that have been implicated in the control of apoptosis (36) and genetic experiments (18,24,48) raise the possibility that modulation of C-RAF kinase activity in survival depends on interaction with a different set of proteins, including Bcl-2 and Bag1 (11,12,43,44). A-RAF, the least well-characterized member of the family, appears to have the lowest specific activity for MEK (32, 51), although it clearly functions as a transforming gene and activates the mitogenic cascade when overexpressed in an activated form (17, 41). Moreover, like B-and C-RAF, A-RAF activation is coupled to stimulation of growth factor receptors such as nerve growth factor and epidermal growth factor receptors and expression of activated variants of all three isozymes causes differentiation and neurite formation in PC12 pheochromocytoma cells (47).Before determination of differentiated cell lineages in midgestation, C-RAF alone can fully compensate B-RAF function and vice versa (18,24,49,50). Double knockout experiments demonstrate that A-RAF alone cannot compensate B-...

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