Cell Mechanosensing Project, International Cooperative Research Project/Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Nagoya 466-8550, Japan; and.
Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9810-5. doi: 10.1073/pnas.1321451111. Epub 2014 Jun 23.
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.
肌动蛋白解聚因子（ADF）/丝切蛋白是一种普遍存在于真核生物中的肌动蛋白丝断裂蛋白，调节肌动蛋白动力学。丝切蛋白与肌动蛋白丝的协同结合对于丝切蛋白浓度依赖性的肌动蛋白动力学的非传统调节至关重要。在这项研究中，使用单分子成像技术直接评估了丝切蛋白与肌动蛋白丝协同结合的动力学参数。当丝切蛋白浓度在 30 nM 至 1 µM 范围内时，丝切蛋白与肌动蛋白丝结合的上结合速率估计为 0.06 µM(-1)⋅s(-1)。丝切蛋白结合的停留时间直方图呈指数衰减，给出了 0.6 s(-1)的离解速率。在长时间的丝切蛋白结合事件（>0.4 s）中，在初始结合位点附近观察到额外的丝切蛋白结合。这些事件的上结合速率比非连续结合高 2.3 倍。对丝切蛋白结合位置的超分辨率图像分析表明，上结合速率增强发生在原始结合事件的 65nm 范围内。相比之下，丝切蛋白的离解速率不受预结合丝切蛋白的影响。降低温度或增加测试溶液的粘度都不会改变结合的上结合速率、离解速率或协同参数（ω）。这些结果表明，丝切蛋白结合增强了初始结合位点附近（约 24 个亚基）额外的丝切蛋白结合，但不影响离解速率，这可能是丝切蛋白与肌动蛋白丝协同结合的分子机制。
