The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.

Background: Spire is a WH2 domain-containing protein implicated in actin nucleation and critical to oogenesis. Results: Spire rapidly depolymerizes actin filaments by combining monomer sequestration with weak filament severing, and it nucleates new filaments. Conclusion: This shows functional and structural variations among actin complexes with Spire. Significance: Spire-actin structures and actin remodeling by Spir are more complex than originally imagined.

Cofilin is a member of the actin depolymerizing factor (ADF)/cofilin family of proteins. It plays a key role in actin dynamics by promoting disassembly and assembly of actin filaments. Upon its binding, cofilin has been shown to bridge two adjacent protomers in filamentous actin (F-actin) and promote the displacement and disordering of subdomain 2 of actin. Here, we present evidence for cofilin promoting a new structural change in the actin filament, as detected via a switch in cross-linking sites. Benzophenone-4-maleimide, which normally forms intramolecular cross-linking in F-actin, cross-links F-actin intermolecularly upon cofilin binding. We mapped the cross-linking sites and found that in the absence of cofilin intramolecular cross-linking occurred between residues Cys374 and Asp11. In contrast, cofilin shifts the cross-linking by this reagent to intermolecular, between residue Cys374, located within subdomain 1 of the upper protomer, and Met44, located in subdomain 2 of the lower protomer. The intermolecular cross-linking of F-actin slows the rate of cofilin dissociation from the filaments and decreases the effect of ionic strength on cofilin−actin binding. These results are consistent with a significant role of filament flexibility in cofilin−actin interactions.

Actin cytoskeleton remodeling is crucial for many vital cellular processes including division, growth, and motility. The actin regulatory protein cofilin contributes to actin remodeling dynamics by creating new filament ends and replenishing the actin monomer pool. Cooperative cofilin binding introduces a topological and mechanical asymmetry at boundaries of bare and cofilin decorated filament segments, which is proposed to facilitate filament severing. Cofilin binding to actin filaments is regulated by phosphorylation. However, the extent to which phosphorylation modulates human cofilin binding cooperativity and severing has not been systematically evaluated. We utilize fluorescence spectroscopy and an Ising model with cooperative nearest-neighbor interactions to quantitate wild-type and phosphomimetic mutant (S3D) human cofilin binding to actin filaments. Modulation of filament mechanics and severing upon cofilin binding is assessed by fluorescence microscopy. The S3D binds filaments with weaker intrinsic binding affinity, yet cooperative interactions are comparable to wild-type cofilin. Preliminary data suggest the phosphorylation mimic mutation also impacts the ability of cofilin to modulate filament mechanics and severing activity.