Despite the wealth of different actin structures formed, only two actin nucleation factors are well established in vertebrates: the Arp2/3 complex and formins. Here, we describe a further nucleator, cordon-bleu (Cobl). Cobl is a brain-enriched protein using three Wiskott-Aldrich syndrome protein homology 2 (WH2) domains for actin binding. Cobl promotes nonbundled, unbranched filaments. Filament formation relies on barbed-end growth and requires all three Cobl WH2 domains and the extended linker L2. We suggest that the nucleation power of Cobl is based on the assembly of three actin monomers in cross-filament orientation. Cobl localizes to sites of high actin dynamics and modulates cell morphology. In neurons, induction of both neurites and neurite branching is dramatically increased by Cobl expression-effects that critically depend on Cobl's actin nucleation ability. Correspondingly, Cobl depletion results in decreased dendritic arborization. Thus, Cobl is an actin nucleator controlling neuronal morphology and development.

Spatial control of cortical actin nucleation is indispensable for proper establishment and plasticity of cell morphology. Cobl is a novel WH2 domain-based actin nucleator. The cellular coordination of Cobl's nucleation activity, however, has remained elusive. Here, we reveal that Cobl's cellular functions are dependent on syndapin. Cobl/syndapin complexes form in vivo, as demonstrated by colocalization, coimmunoprecipitation and subcellular recruitment studies. In vitro reconstitutions and subcellular fractionations demonstrate that, via its lipid-binding Fer/CIP4 Homology (FCH)-Bin/Amphiphysin/Rvs (F-BAR) domain, syndapin recruits Cobl to membranes. Consistently, syndapin I RNAi impairs cortical localization of Cobl. Further functional studies in neurons show that Cobl and syndapin I work together in dendritic arbor development. Importantly, both proteins are crucial for dendritogenesis. Cobl-mediated functions in neuromorphogenesis critically rely on syndapin I and interestingly also on Arp3. Endogenous Cobl, syndapin I and the Arp2/3 complex activator and syndapin-binding partner N-WASP were present in one complex, as demonstrated by coimmunoprecipitations. Together, these data provide detailed insights into the molecular basis for Cobl-mediated functions and reveal that different actin nucleators are functionally intertwined by syndapin I during neuromorphogenesis.

Polymerization and organization of actin into complex superstructures, including those found in dendritic spines, is indispensable for structure and function of neuronal networks. Here we show that the filamentous actin (F-actin)-binding protein 1 (Abp1), which controls Arp2/3 complex-mediated actin nucleation and binds to postsynaptic scaffold proteins of the ProSAP (proline-rich synapse-associated protein 1)/Shank family, has a profound impact on synaptic organization. Overexpression of the two Abp1 F-actin-binding domains increases the length of thin, filopodia-like and mushroom-type spines but dramatically reduces mushroom spine density, attributable to lack of the Abp1 Src homology 3 (SH3) domain. In contrast, overexpression of full-length Abp1 increases mushroom spine and synapse density. The SH3 domain alone has a dominant-negative effect on mushroom spines, whereas the density of filopodia and thin, immature spines remains unchanged. This suggests that both actin-binding and SH3 domain interactions are crucial for the role of Abp1 in spine maturation. Indeed, Abp1 knockdown significantly reduces mushroom spine and synapse density. Abp1 hereby works in close conjunction with ProSAP1/Shank2 and ProSAP2/Shank3, because Abp1 effects were suppressed by ProSAP2 RNA interference and the ProSAP/ Shank-induced increase of spine head width is further promoted by Abp1 cooverexpression and reduced on Abp1 knockdown. Also, interfering with the formation of functional Abp1-ProSAP protein complexes prevents ProSAP-mediated spine head extension. Spine head extension furthermore depends on local Arp2/3 complex-mediated actin polymerization, which is controlled by Abp1 via the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Abp1 thus plays an important role in the formation and morphology control of synapses by making a required functional connection between postsynaptic density components and postsynaptic actin dynamics.