Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.

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The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain. Consistently, PRMT2 phenocopied Cobl functions in both gain- and loss-of-function studies. Both PRMT2- and Cobl-promoted dendritogenesis relied on methylation. PRMT2 effects require both its catalytic domain and SH3 domain. Cobl-mediated dendritic arborization required PRMT2, complex formation with PRMT2, and PRMT2's catalytic activity. Mechanistic studies reveal that Cobl methylation is key for Cobl actin binding. Therefore, arginine methylation is a regulatory mechanism reaching beyond controlling nuclear processes. It also controls a major, cytosolic, cytoskeletal component shaping neuronal cells.