Haimo L T, Telzer B R, Rosenbaum J L
Proc Natl Acad Sci U S A. 1979 Nov;76(11):5759-63. doi: 10.1073/pnas.76.11.5759.
Dynein isolated from Chlamydomonas flagellar axonemes binds to microtubules assembled in vitro from 6S brain tubulin dimers. The dynein arms bind periodically along the length of the microtubules with a center-to-center spacing of 24 nm, equal to the periodicity of dynein arms on intact axonemes. The arms project from the in vitro assembled microtubules at an angle of approximately 55 degrees, thereby defining microtubule polarity. Dynein cosediments with microtubules through a sucrose gradient, as demonstrated by electron microscopy, gel electrophoresis, and ATPase analysis. In addition, dynein induces crossbridging between adjacent microtubules. Darkfield microscopy reveals that microtubules containing dynein are aggregated into large bundles; electron microscopy indicates that microtubules of the same polarity are crossbridged by a regular array of arms. Viewed by darkfield microscopy, addition of ATP to crossbridged microtubules causes their disaggregation; electron microscopy shows that the majority of these microtubules are no longer crossbridged. These observations are applicable to the determination of microtubule polarity and directionality of microtubule assembly in situ and suggest a role for dynein in cytoplasmic microtubule-based cellular movements.
从衣藻鞭毛轴丝中分离出的动力蛋白可与由6S脑微管蛋白二聚体在体外组装而成的微管结合。动力蛋白臂沿着微管长度呈周期性结合，中心间距为24纳米，这与完整轴丝上动力蛋白臂的周期性相同。动力蛋白臂以约55度的角度从体外组装的微管伸出，从而确定微管的极性。通过蔗糖梯度离心，动力蛋白与微管共同沉降，这一点通过电子显微镜、凝胶电泳和ATP酶分析得到了证实。此外，动力蛋白可诱导相邻微管之间形成交联桥。暗视野显微镜观察显示，含有动力蛋白的微管聚集成大束；电子显微镜表明，相同极性的微管由规则排列的臂交联在一起。在暗视野显微镜下观察到，向交联的微管中添加ATP会导致它们解聚；电子显微镜显示，这些微管中的大多数不再交联。这些观察结果适用于原位确定微管的极性和微管组装的方向性，并提示动力蛋白在基于细胞质微管的细胞运动中发挥作用。
