Hisanaga S, Hirokawa N
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
J Mol Biol. 1987 Jun 20;195(4):919-27. doi: 10.1016/0022-2836(87)90495-5.
The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.
采用快速冷冻、深度蚀刻技术研究了细胞质动力蛋白分子的亚结构。从海胆光棘球海胆的卵中纯化得到的12S形式的细胞质动力蛋白由一条单一的高分子量多肽组成。喷镀在云母表面或快速冷冻在云母片上的细胞质动力蛋白的旋转阴影图像显示为单头分子，这与海胆精子鞭毛21S动力蛋白的双头分子形成对比。通过快速冷冻深度蚀刻后的旋转阴影观察到了更详细的亚结构。细胞质动力蛋白由一个头部和一个柄部组成。头部呈梨形（16nm×11nm），比21S动力蛋白的梨形头部（18nm×14nm）略小。柄部的形态不规则，其表观长度从0到32nm不等。通过负染色观察了溶液中细胞质动力蛋白与脑微管的结合情况，同时也用快速冷冻、深度蚀刻法检查了沉淀物中的结合情况。两种方法都揭示了存在两种微管，一种是完全被装饰的微管，另一种是未被装饰的微管。与微管结合的细胞质动力蛋白也呈现为球状颗粒。细胞质动力蛋白虽然似乎协同结合到微管上，但并未形成21S动力蛋白所观察到的周期性结合或交叉桥。
