The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

Abstract

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.

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

“…Cell culture, transfection, coprecipitation with calmodulin and detection of associated proteins were conducted as described previously (Hou et al, 2015 PLoS Biol.). Briefly, HEK293 cells were cultured at 37°C under 5% CO2 in DMEM medium (Gibco Life technologies) additionally supplied with 10% fetal bovine serum, 0.1 mM gentamicin and 1x penicillin (100 units/ml)-streptomycin (100 μg/ml).…”

YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis In Vivo

“…Genes encoding proteins that regulate the actin cytoskeleton were also newly accessible YAP5SA targets (Morikawa et al, 2015). Pak3 encodes a protein that regulates actin cytoskeleton by modulating small GTPase activity (Felix et al, 2015;Hiramoto-Yamaki et al, 2010), whereas Cobl and Fmn1 encode actin nucleators (Hou et al, 2015;Li et al, 2015b) (Figures 5F and S5; Table S1).…”

It takes a village to raise a branch: Cellular mechanisms of the initiation of axon collateral branches

“…Therefore, additional actin filament nucleators may also operate in actin patches to generate the mother filaments that the Arp2/3 complex subsequently binds (Figure 2B). The actin nucleation factor cordon-bleu is a likely candidate as it positively contributes to the formation of axon collateral branches in hippocampal neurons, and can cooperate with Arp2/3 in the formation of actin filaments (Ahuja et al, 2007; Hou et al, 2015). Nevertheless, the specific role of cordon bleu in actin patch and filopodia initiation during axonal branching needs further studies.…”