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Development of a New Optical Microscope System, which Resolves Motion of Motor Protein Molecules Less than Lnm on the Sub-Millisecond Time Scale.

开发新型光学显微镜系统，可在亚毫秒时间尺度上解析小于 Lnm 的运动蛋白分子的运动。

基本信息

批准号：
63880034
负责人：
YANAGIDA Toshio
金额：
$ 9.66万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B).
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

We have developed a new system for measuring the force produced by a small number (1-100) of myosin molecules interacting with a single actin filament in vitro. The technique can resolve the motion of actin filaments less than 1 nm on the sub-millisecond time scale and thus detect fluctuations of force due to the individual molecular events. Development of this new system followed the design of techniques for force measurement in vitro motility assay by manipulating single actin filaments attached to glass microneedles under a fluorescence microscope.Analysis of force fluctuations (noise) is a very powerful approach toward determining kinetic characteristics of individual myosin heads interacting with actin, especially when the number of interactions is small as in the present conditions. Under isometric conditions, we have observed large force fluctuations similar to membrane current fluctuations due to channel gating in electrophysiological systems with small numbers of channels.The … More force fluctuations in the isometric condition are consistent with models incorporating stochastic and independent molecular events. Both a simplified, two state, ON-OFF model and the Huxley (1957) model simulate the amplitude and frequency spectrum of the observed cross-bridge noise. Using these models we determined the ON and OFF rates and the force generated by single myosin heads. The data are compatible with one fundamental mechanical interaction for each ATPase cycle.On the other hand, when the actin filaments actively slide at velocities greater than -1 mum/s, fluctuations of force are much smaller. The data analysis indicates that myosin produces an almost constant force for greater than 90 % of the ATPase cycle time. This result indicates that during sliding, for each ATPase cycle there are many force-generating mechanical interactions between actin and myosin.Thus, the present system allows us to directly examine how the ATPase cycle is coupled to the mechanical interactions of individual myosin heads with actin in vitro. These conclusions also apply directly to the molecular mechanism of muscle contraction. Less

我们开发了一种新的系统，用于在体外测量少量(1-100)肌球蛋白分子与单个肌动蛋白细丝相互作用所产生的力。这项技术可以在亚毫秒的时间尺度上分辨出小于1纳米的肌动蛋白细丝的运动，从而检测到由于单个分子事件而引起的力的波动。这一新系统的开发遵循了通过在荧光显微镜下操纵附着在玻璃微针上的单个肌动蛋白细丝来进行体外动力测试的技术设计。力波动(噪声)的分析是确定单个肌球蛋白头部与肌动蛋白相互作用的动力学特征的非常有效的方法，特别是当相互作用的次数很少时，如在目前的条件下。在等长条件下，我们观察到了在通道数较少的电生理系统中，由于通道选通而引起的类似于膜电流波动的大的力波动。…在等轴测条件下，更多的力波动与包含随机和独立分子事件的模型一致。简化的两态开关模型和Huxley(1957)模型都模拟了观测到的跨桥噪声的幅度和频谱。利用这些模型，我们确定了单个肌球蛋白头的开启和关闭速率以及所产生的力。另一方面，当肌动蛋白细丝以大于-1微米/S的速度主动滑动时，力的波动要小得多。数据分析表明，肌球蛋白在超过90%的ATPase周期时间内产生几乎恒定的力。这一结果表明，在滑动过程中，对于每个ATPase循环，肌动蛋白和肌球蛋白之间存在许多产生力的机械相互作用。因此，本系统允许我们直接研究ATPase循环是如何与体外单个肌球蛋白头部与肌动蛋白的机械作用相耦合的。这些结论也直接适用于肌肉收缩的分子机制。较少