Cell migration, which is central to many biological processes including wound healing and cancer progression, is sensitive to environmental stiffness, and many cell types exhibit a stiffness optimum, at which migration is maximal. Here we present a cell migration simulator that predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction is verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ∼1 kPa) and U251 glioma cells (optimum ∼100 kPa). In addition, the model predicts, and experiments confirm, that the stiffness optimum of U251 glioma cell migration, morphology and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions.

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 µm, corresponding to a flexural rigidity of 0.040 pN µm 2 . Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 µm and the filament flexural rigidity to 0.0091 pN µm 2 . That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and forceproducing properties of cells can be modulated by cofilin activity.

Summary Cell motility driven by actin filament assembly demands the spatial and temporal coordination of numerous regulatory Actin Binding Proteins (ABPs) [1], many of which bind with affinities and kinetics that depend on the chemical state (ATP, ADP-Pi or ADP) of actin filament subunits. ADF/cofilin, one of three ABPs that precisely choreograph actin assembly and organization into “comet-tails” that drive motility in reconstituted in vitro systems [2], binds and stochastically severs “aged” ADP actin filament segments of de novo growing actin filaments [3]. Severing increases the density of filament ends from which subunits can add and dissociate, thereby increasing overall actin filament assembly dynamics. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments as well as ADF/cofilin severing limits the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled ADF/cofilin that retains actin filament binding and severing activities. Since ADF/cofilin binding depends strongly on the actin-bound nucleotide direct visualization of fluorescent ADF/cofilin binding serves as a marker of the actin filament nucleotide state and permits assessment of the “ATP/ADP-Pi cap” length of individual actin filaments during assembly and elongation. Bound ADF/cofilin allosterically accelerates Pi release from unoccupied filament subunits, which shortens the filament ATP/ADP-Pi cap length by nearly an order of magnitude. Rapid elongation far exceeds ADF/cofilin-acceleration of Pi release under in vivo conditions; thereby filament barbed end capping is required for efficient ADF/cofilin binding and severing. Real time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and ADF/cofilin decorated filament segments, thereby controlling the overall filament length depending on the ADF/cofilin activity and filament binding density.

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.