Structural and functional insights into the Spir/formin actin nucleator complex.

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.

Molecular basis of actin nucleation factor cooperativity: crystal structure of the Spir-1 kinase non-catalytic C-lobe domain (KIND)•formin-2 formin SPIR interaction motif (FSI) complex.

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.

Cortical migration defects in mice expressing A-RAF from the B-RAF locus.

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.

Enhanced fear expression in Spir-1 actin organizer mutant mice.

Spir proteins nucleate actin filaments at vesicle membranes and facilitate intracellular transport processes. The mammalian genome encodes two Spir proteins, namely Spir-1 and Spir-2. While the mouse spir-2 gene has a rather broad expression pattern, high levels of spir-1 expression are restricted to the nervous system, oocytes, and testis. Spir-1 mutant mice generated by a gene trap method have been employed to address Spir-1 function during mouse development and in adult mouse tissues, with a specific emphasis on viability, reproduction, and the nervous system. The gene trap cassette disrupts Spir-1 expression between the N-terminal KIND domain and the WH2 domain cluster. Spir-1 mutant mice are viable and were born in a Mendelian ratio. In accordance with the redundant function of Spir-1 and Spir-2 in oocyte maturation, spir-1 mutant mice are fertile. The overall brain anatomy of spir-1 mutant mice is not altered and visual and motor functions of the mice remain normal. Microscopic analysis shows a slight reduction in the number of dendritic spines on cortical neurons. Detailed behavioral studies of the spir-1 mutant mice, however, unveiled a very specific and highly significant phenotype in terms of fear learning in male mice. In contextual and cued fear conditioning experiments the male spir-1 mutant mice display increased fear memory when compared to their control littermates. Our data point toward a particular function of the vesicle associated Spir-1 actin organizer in neuronal circuits determining fear behavior.

Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division.

Oocytes 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.

Expression patterns of the mouse Spir-2 actin nucleator.

Spir proteins are the founding members of the novel class of WH2 domain containing actin nucleation factors. They initiate actin polymerization by binding of actin monomers to four WH2 domains in the central part of the proteins. Despite their ability to nucleate actin polymerization in vitro by themselves, Spir proteins form a regulatory complex with the distinct actin nucleators of the formin subgroup of formins. The mammalian genome encodes two spir genes, spir-1 and spir-2. The corresponding proteins have an identical structural array and share a high degree of homology. Here, we have addressed the yet unknown expression of the mouse spir-2 gene. Northern blot analysis revealed that the spir-2 gene is expressed as a single mRNA. During embryogenesis in situ hybridizations show spir-2 to be expressed in the developing nervous system and intestine. In adult mouse tissues highest expression of spir-2 was detected in the epithelial cells of the digestive tract and in neuronal cells of the nervous system. High expression was also detected in testical spermatocytes. In contrast to the restricted expression of the mouse spir-1 gene, which is mainly found in the nervous system, our data presented here show a distinct and broader expression pattern of the spir-2 gene and by this support a more general cell biological function of the novel actin nucleators.