Fluctuation analysis of actin in vitro sliding along native thick filaments from molluscan smooth muscles

1)我们从软体动物平滑肌中纯化了长肌球蛋白粗丝，其中肌球蛋白马达定向良好，并且我们记录了许多肌动蛋白丝在体外沿着沿着单个肌球蛋白粗丝的不同长度的滑动运动。我们用五种不同的肌球蛋白厚度的应变仪获得了良好的记录。2）我们通过计算给定时间段内肌动蛋白丝滑动距离的方差来分析肌动蛋白丝滑动距离的波动。该分析产生每个肌动蛋白丝的有效扩散系数Din的值。然后我们研究了Dm对肌动蛋白丝长度的依赖性，发现Dm不依赖于肌动蛋白的长度，并且是恒定的，就像以前在体外随机取向的马达蛋白一样。3）通常认为，马达蛋白在驱动细胞骨架丝单向滑动中的作用是统计独立和随机的。如果是这种情况，我们可以预期Dm与肌动蛋白丝长度的倒数成正比，这是中心极限定理的直接结果。通过用Huxley 1957年的“弹簧肌球蛋白”模型进行计算机模拟，我们证实了这一理论预测，即如果运动动作是随机的和统计独立的，则滑动距离的波动与细丝长度的倒数成比例。上述Din不依赖于肌动蛋白丝长度的实验发现表明，这种信念在体外的情况下并不成立。5）因此，我们认为肌球蛋白马达在产生肌动蛋白丝单向滑动运动中的作用不是随机的，它们的作用中一定存在着协同性。我们目前正在研究可能的机制，通过计算机模拟的合作。

1) We purified long myosin thick filaments from molluscan smooth muscles, in which myosin motors are well oriented, and we recorded sliding movements of many actin filaments with various lengths along individual myosin thick filaments in vitro. We obtained good recordings with five different myosin thick filaments.2) We analyzed the fluctuation of the sliding distance of actin filaments, by calculating the variance of the sliding distance for a given period of time. The analysis yields a value of an effective diffusion coefficient, Din, for each actin filament. We then studied dependence of Dm on the actin filament length, and found that Dm does not depend on the actin length and is constant, as was previously found with randomly oriented motor proteins in vitro.3) It is often believed that the actions of motor proteins in driving a cytoskeletal filament to slide unidirectionally are statistically independent and random. If this is the case, we can expect that Dm is proportional to the inverse of the actin filament length, which is a direct consequence of the central limit theorem. By computer simulation with Huxley's 1957 model of 'spring myosins' we confirmed this theoretical prediction that the fluctuation of sliding distance is proportional to the inverse of the filament length if the motor actions are random and statistically independent.4) However, the above experimental finding that Din does not depend on the actin filament length indicates that this belief does not hold in the case of the in vitro sliding movement generation by protein motors, and instead indicates that there is a cooperativity in the mechanism of the directional sliding movement generation by motor proteins.5) We therefore conclude that the actions of myosin motors in generating the unidirectional sliding movement of actin filaments are not random and there must be cooperativity in their actions. We are currently studying possible mechanisms of the cooperativity by computer simulation.