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

肌动蛋白沿着软体动物平滑肌天然粗丝滑动的体外波动分析

基本信息

批准号：
09480178
负责人：
TAWADA Katsuhisa
金额：
$ 8.7万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

1)我们从软体动物肌肉中提纯了肌球蛋白粗丝，肌球蛋白马达定向良好，并在体外记录了许多不同长度的肌球蛋白粗丝沿着单个肌球蛋白粗丝的滑动运动。我们用五种不同的肌球蛋白粗丝获得了良好的记录。2)通过计算一段时间内肌球蛋白细丝滑动距离的方差，分析了肌动蛋白细丝滑动距离的波动。该分析产生了每个肌动蛋白细丝的有效扩散系数Din的值。然后我们研究了DM对肌动蛋白细丝长度的依赖关系，发现DM不依赖于肌动蛋白的长度并且是恒定的，就像以前在体外发现的随机取向的马达蛋白一样。3)人们通常认为马达蛋白在驱动细胞骨架细丝单向滑动方面的动作在统计上是独立的和随机的。如果是这种情况，我们可以预期Dm与肌动蛋白细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，即如果运动是随机的且统计上独立的，则滑动距离的波动与细丝长度的反比成正比。4)然而，上述实验发现Din不依赖于肌动蛋白细丝长度，这一信念在蛋白质马达产生的体外滑动运动的情况下不成立，5)因此，我们得出结论，肌球蛋白马达在产生肌动蛋白细丝的单向滑动运动中的动作不是随机的，它们的动作中一定存在协同作用。我们目前正在通过计算机模拟来研究这种协同性的可能机制。