Cofilin-Linked Changes in Actin Filament Flexibility Promote Severing

Abstract

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.

Site-specific cation release drives actin filament severing by vertebrate cofilin

“…The average steady-state filament length ( Fig. 2 C and D) confirms that WT yeast actin filaments are weakly severed by vertebrate cofilin, whereas A167E yeast actin filaments partially decorated with cofilin are severed more efficiently than bare or saturated filaments, similar to vertebrate actin filaments (12,16).…”

“…2A and Fig. S1) despite binding with tight affinity (K d <100 nM) (12), comparable to the affinity of vertebrate cofilin binding to A167E yeast actin filaments (Fig. S2).…”

“…A lower-resolution (∼20 Å) reconstruction of cofilactin filaments under low-salt conditions did not reveal obvious differences in cofilin binding modes or conformations, and the filament pitch and rise were indistinguishable between low-and high-salt conditions. (12), yielding severing rate constants at boundaries relative to spontaneous fragmentation rate of bare actin filaments: k′ sc = 0.9 (± 0.7) and 8.3 (± 3.2) for WT and A167E yeast actin filaments, respectively. Severing seems to be more efficient in real-time assays, presumably owing to inhibition of filament reannealing by flow.…”

“…Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10-13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21).…”

“…Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10-13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).…”