title: 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 single-molecule 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 off-rate 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 on-rates, 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.
authors: Cell Mechanosensing Project, International Cooperative Research Project/Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Nagoya 466-8550, Japan; and.
authors: Kimihide
authors: K
authors: Hayakawa
authors: Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors: Shotaro
authors: S
authors: Sakakibara
authors: Cell Mechanosensing Project, International Cooperative Research Project/Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Nagoya 466-8550, Japan; andDepartment of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors: Masahiro
authors: M
authors: Sokabe
authors: Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan tatsumi@med.nagoya-u.ac.jp.
authors: Hitoshi
authors: H
authors: Tatsumi
mesh_terms: D000199:Actins / Q000378:metabolism; D051338:Cofilin 1 / Q000378:metabolism*; D051340:Cofilin 2 / Q000378:metabolism*; D051345:Destrin / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013696:Temperature; D014783:Viscosity
chemical_list: D000199:Actins; D051338:Cofilin 1; D051340:Cofilin 2; D051345:Destrin
keywords: cooperativity; fluctuations; non--nearest-neighbor; non-Arrhenius; superresolution