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.

Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function, filament assembly and fragmentation, and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features, including the subunit dimensions, interaction energies, helicity, and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values, demonstrating the capacity of the model to assess the mechanical properties of actin filaments, including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong, with a persistence length of 0.15-0.4 μm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling, with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, L, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: -2/3). We also showed that the average subunit residence time inside the filament, T, depends on the actin monomer (power law: -1/6) and ADF/cofilin (power law: -2/3) concentrations. In addition, filament length fluctuations are approximately 20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P(i) versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.

The regulatory protein, cofilin, severs actin filaments and increases the number of ends from which subunits add and dissociate. Structural and biochemical analyses demonstrate that cofilin binding alters the conformation and mechanics of actin filaments such that cofilin-decorated filaments are ~20-fold more compliant in bend and twist than native actin filaments. Equilibrium and kinetic binding models as well as direct visualization of cofilin binding to filaments favor a mechanism in which severing occurs at or near boundaries of bare and cofilin-decorated segments. It is hypothesized that shear stress associated with conformational fluctuations accumulates locally at boundaries of mechanical asymmetry, thereby leading to preferential severing at junctions of bare and cofilin-decorated segments. In this work, we evaluate if mechanical and confor-mational periodicity in filaments promotes stress accumulation at junctions of asymmetry (i.e. boundaries). We have derived mathematical expressions of the actin filament elastic free energy, accounting for contributions from bending, twisting and twist-bend coupling, and used a computational modeling approach to evaluate the distribution of energy and stress of model filaments strained by external mechanical (buckling or torque) loads applied to filament ends. Our results indicate that mechanical asymmetry introduced by cofilin binding promotes the accumulation of shear stress at boundaries between bare and cofilin-decorated segments that likely increases the probability of failure (i.e. severing) under active or passive, thermal deformation, analogous to the fracture of some non-protein materials. Elastic coupling between twisting and bending is critical for stress accumulation at boundaries. Proteins of the ADF/cofilin family are vital regulators of the actin cytoskeleton in eukaryotes. Binding of cofilin results in dramatic reorganization of the F-ac-tin structure and potentiates filament severing, and its accelerated depolymer-ization from the pointed end. Although the X-ray structure of monomeric actin with cofilin-homology domain of twinfilin has been recently solved, F-ac-tin evades crystallization and therefore the analysis of cofilin interaction with F-actin at the atomic level calls for alternative approaches. While considerable insight into the cofilin F-actin complex has been gained through chemical cross-linking and radiolytic footprinting, these approaches were unable to generate high resolution information on this interaction While considerable insight into the cofilin F-actin complex has been gained through chemical cross-linking and radiolytic footprinting, novel approaches are still desirable. Here we demonstrate that Fast-MAS Solid State NMR is a high sensitivity approach to studying this system. Isotopically labeled S. cerevisieae yeast cofilin, in complex with polymerized yeast actin, allows for an atomic resolution view of co-filin within the complex. Intramolecular conformational changes occurring in cofilin upon binding to actin can be deduced...