Mutagenetic and electron microscopy analysis of actin filament severing by Cordon‐Bleu, a WH2 domain protein

Abstract

Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer » of actin assembly, inducing fast turnover of actin filaments and oscillatory polymerization regime via nucleation, severing, and rapid depolymerization activities. Cobl is the most efficient severer of actin filaments characterized so far. To understand which primary sequence elements determine the filament severing activity of the WH2 repeats, here we combine a mutagenetic/domain swapping approach of the minimal fully active Cobl-KAB construct, which comprises the lysine rich region K preceding the two first WH2 domains A and B. The mutated Cobl constructs display variable loss of the original filament nucleating activities of native Cobl-KAB, without any strict correlation with a loss in actin binding, which emphasizes the functional importance of the electrostatic environment of WH2 domains. Filament severing displayed the greatest stringency and was abolished in all mutated forms of Cobl-KAB. Filament severing and re-annealing by Cobl-KAB, which is key in its rapid remodeling of a population of actin filaments, and most likely responsible for its function in ciliogenesis, was analyzed by electron microscopy in comparison with Spire and ADF.

Enhanced Depolymerization of Actin Filaments by ADF/Cofilin and Monomer Funneling by Capping Protein Cooperate to Accelerate Barbed-End Growth

“…Upon exposure to ADF, we observed both filament severing as well as faster pointed-end depolymerization (between consecutive severing events; Figure 1B). The frequency of severing increased with ADF concentration, as seen previously [22, 36]. Figure 1E shows that 50% of filaments (mean length = 10 μm) have severed at least once at t 1/2  = 40 s in the presence of 5 μM ADF, consistent with the scheme F → 2F and a first-order fragmentation rate constant of ln(2)/t 1/2  = 0.017 s −1 for a 10-μm-long filament (see Movie S4).…”

Direct visualization of how Actin Depolymerizing Factor’s filament severing and depolymerization synergizes with Capping Protein's "monomer funneling" to promote rapid polarized growth of actin filaments

“…Upon exposure to ADF, we observed both severing of filaments and faster pointed-end depolymerization between consecutive severing events ( Figure 1b). The frequency of severing increased with ADF concentration, as seen previously [18,34]. Figure 1e shows that 50% of filaments (mean length = 10µm) have severed once at t1/2 = 40s in the presence of 5 µM ADF, consistent with the scheme F -> 2F and a first order fragmentation rate constant of ln(2)/t1/2 = 0.017 s -1 for a 10 µm long filament.…”

Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains

“…Adjacent to the first WH2 domain is a lysine-rich patch (Figure 2A) that is necessary for both nucleating and severing actin filaments (Husson et al. , 2011; Jiao et al. , 2014).…”

“…Recent work also suggests that WH2 domains can be powerful severing agents (Husson et al. , 2011; Jiao et al. , 2014).…”