Single-molecule imaging and kinetic analysis of cooperative cofilin–actin filament interactions

Abstract

The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics. The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin. In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a singlemolecule imaging technique. The on-rate of cofilin binding to the actin filament was estimated to be 0.06 μM −1 ·s −1 when the cofilin concentration was in the range of 30 nM to 1 μM. A dwell time histogram of cofilin bindings decays exponentially to give an offrate of 0.6 s −1 . During long-term cofilin binding events (>0.4 s), additional cofilin bindings were observed in the vicinity of the initial binding site. The on-rate for these events was 2.3-fold higher than that for noncontiguous bindings. Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event. By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin. Neither decreasing the temperature nor increasing the viscosity of the test solution altered the onrates, off-rates, or the cooperative parameter (ω) of the binding. These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca. 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments.cooperativity | fluctuations | non-nearest-neighbor | non-Arrhenius | superresolution C ofilin (1, 2) induces actin disassembly by severing actin filaments at concentrations below the equilibrium dissociation constant, whereas it facilitates actin nucleation at higher concentrations (3). This concentration-dependent modulation of cofilin activity is presumably due to its cooperative binding to actin filaments (4). In the wider sense, understanding the cooperative interactions between proteins is essential for understanding the regulation of enzymes (5, 6). However, the dynamic protein interactions that endow proteins with cooperativity in solution have not been directly imaged and analyzed. In this study, we focused on the molecular mechanism underlying the cooperative binding of cofilin to actin filaments using high-resolution optical microscopy.There is a general agreement that the binding of cofilin to actin filaments changes the conformation of the actin filaments. Electron microscopic observations reveal that the binding of cofilin to actin filaments increases their twist (4, 7). Biochemical analyses of the kinetics of cofilin binding to actin filaments suggest that the conformational changes induced by cofilin binding affect further binding of cofilin to the actin filament (8). Differential scanning calorimetric study of cofilin-actin complexes indicates that the allosteric destabilization of ...

Actin Mechanics and Fragmentation

“…Vertebrate cofilin binding is positively cooperative (43). Association is far below the diffusion limit for encounter (45) and limited by filament shape fluctuations (45,46), suggesting opportunistic binding to compliant filament segments (45). Severing occurs preferentially at or near boundaries (e.g.…”

The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments

“…Estimates of the length over which cofilininduced conformational changes and cooperative binding interactions propagate along actin vary, ranging from N = 1-2 up to N > 100 subunits (3,5,(12)(13)(14)(15)(16)(17)(18)(19)(20). Equilibrium (3,6,12,21) and transient kinetic (12,14) binding data are well described by models invoking positive cooperativity between nearest neighbors (N = 1-2).…”

“…In contrast, differential scanning calorimetric (13) and spectroscopic lifetime (15) measurements estimate allosteric propagation of changes in structure, stability, and/or dynamics over N > 100 subunits. More recently, a singlemolecule TIRF study measured positive cooperative binding interactions that propagated exponentially with a decay length of N ~24 subunits (18), and atomic force microscopic imaging directly observed a change in the crossover distance of N ~14 bare actin subunits towards the pointed-end side of the boundary, but no propagation in the bare actin subunits towards the barbedend side of the boundary (8).…”

Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes

“…46 Not only stress fibers but also actin filaments in the lamellipodium may respond to periodic stretching stimuli. In any case, the cytoskeleton is likely to be affected by the stretching and the relaxation via some form of molecular dynamics, such as the binding of cofilin to relaxed actin filaments 50,51 which induces the depolymerization of the filaments or the opening of stretch-activated Ca 2C channels 53 which induces the contraction of actomyosin. To reveal the connection of stress fibers to migration direction preference and what molecular dynamics is triggered by the repeated stretching of keratocytes, the application of repeated stretching to single live keratocytes will be required in future studies.…”

“…[26][27][28][29][30][31][32] The cause of this reaction is thought to be the depolymerization of stress fibers aligned parallel to the direction of stretching by the binding of the actin-severing protein cofilin. 50,51 To test whether the depolymerization of stress fibers in response to the periodic stretching stimuli is required for the directional migration of keratocytes, we treated them with jasplakinolide, which inhibits the depolymerization of stress fibers composed of actin filaments. We then applied periodic stretching stimuli.…”