High-Resolution Structural Aanlyases of Dynein Molecules During Sliding By Quick-Freeze Deep-Etoh Replica Electron Microscopy

本项目的目的是确定细胞质dyncin/微管复合体的三维结构及其在滑动运动中的变化。由于很难获得非常新鲜、活性很高的材料，我们将材料改为海胆精子鞭毛，并研究了轴丝动力蛋白亚种的结构/功能关系。我们还研究了滑动肌球蛋白复合体的三维结构，使用了我们最近根据单分子生理学的概念发展起来的重建方法。以这些研究结果为基础，我们讨论了在dynein/microtuble滑动系统中经常起关键作用的进行性运动的分子机制。肌球蛋白V是最耐人寻味的，因为它有长长的杠杆臂部分，以及它沿着肌动蛋白细丝以36纳米的步幅“行走”的运动。肌球蛋白-V…的这种特征行为More被认为是支持“倾斜杠杆-手臂机制”有效性的最有力证据之一。我们研究了带有截短杠杆臂的肌球蛋白V突变体和具有短臂结构的肌球蛋白VI突变体的行为。有趣的是，它们都显示了肌动蛋白的进行性运动，步长约为36 nm，与原始的肌球蛋白V相同，具有长杠杆臂。我们用电子显微镜检查了这些肌球蛋白头部与肌球蛋白突变体的结合方式，这些突变体的ADP-PI状态非常稳定。令人惊讶的是，我们发现所有这些肌球蛋白头部似乎都以~36 nm的规则间隔与肌动蛋白结合，并且只与肌丝的一侧结合。这些现象很难用“倾斜杠杆-臂假说”来解释，这可能意味着一个被激活的肌球蛋白头部与肌动蛋白结合后，可能会在距离原始部位36 nm的地方引起特殊的高亲和力部位(S)(热点(S))。根据我们的复制品图像，肌球蛋白头部的功能结构大致分为三类：通过50kD以上结构域与肌动蛋白结合的结构，通过较低50kD结构域与肌球蛋白结合的结构，以及通过这两个50kD结构域结合的肌球蛋白头部结构。如果假设具有不同结构的颗粒代表肌球蛋白结构变化的时间进程，我们可能会认为肌球蛋白头部进行肌动蛋白细丝，涉及一定的“摇摆运动”。海胆轴丝的B带成分被证明是dyncin亚种。较少

This project originally aimed to determine three-dimensional (3-D) structure of cytoplasmic dyncin/microtubule complex and its change upon sliding movement. Because of difficulty in obtaining very fresh materials with high activity, we switched the material to sea-urchin sperm flagella and studied the structure/function relationship of axonemal dyncin subspecies. We also investigated the 3-D structure of sliding actomyosin complex, using a reconstruction method which we recently developed according to the concept of "single molecule physiology". With the results of such studies as a basis, we discussed the molecular mechanism of processive motor activity which often seems to play crucial roles in dynein/microotuble sliding systems.Many members of unconventional myosins evoke processive movement of actin filament. Myosin-V is among the most intriguing for its long lever-arm moiety and its "walking" movement with 36 nm steps, along actin filament. Such characteristic behavior of myosin-V … More has been attributed as one of the strongest evidences to support the validity of "tilting lever-arm mechanisms". We examined the behavior of myosin-V mutant with truncated lever-arm and myosin-VI which naturally has short lever-arm moiety. Interestingly, both of them showed processive movement of actin with 〜36 nm step size that is identical to original myosin-V with long lever-arm. We checked the mode of binding of those myosin heads together with dyctyostelium myosin mutant whose ADP-Pi state is extraordinarily stabilized, to actin filament by electron microscopy. Surprisingly, we found that all of those myosin heads seem to bind to actin with 〜36 nm regular intervals, and only to one side of the filament. All of these phenomena are quite difficult to explain by "tilting lever-arm hypothesis" and might suggest the possibility that the binding of one energized myosin head to actin might evoke the special high affinity site(s) (hot-spot(s)) 〜36 nm away from the original site.According to our replica images, the structure of functioning myosin heads was roughly classified into three categories,' those bound to actin through upper 50kD domain, those bound through lower 50kD domain and those bound through both 50kD domains. If the particles with different structures are postulated to represent the time-course of myosin's structural change, we might assume that myosin head proceeds actin filament involving certain "rocking motion".The B-band component of sea urchin axoneme turned out to be a dyncin subspecies. Less