Tsukita S, Tsukita S, Kobayashi T, Matsumoto G
J Cell Biol. 1986 May;102(5):1710-25. doi: 10.1083/jcb.102.5.1710.
In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.
在前一篇论文中（小林，T.，筑田，S.，筑田，S.，山本，Y.，松本，G.，1986，《细胞生物学杂志》，102:1710 - 1725），我们通过生化方法证明了乌贼巨大轴突的轴膜下细胞骨架高度特化，主要由微管蛋白、肌动蛋白、轴突素和一种255 kD的蛋白质组成。在本文中，我们对轴膜下细胞骨架在原位的分子组织进行了形态学分析。对于超薄切片电子显微镜，通过向轴突内灌注含单宁酸的固定剂对轴膜下细胞骨架进行化学固定。采用这种固定方法，超微结构的完整性得到了很好的保存。对于冷冻蚀刻复型电子显微镜，将经轴突内灌注的轴突打开，通过使其内表面接触冷却的铜块（4 K）快速冷冻，从而可以直接立体观察轴膜的细胞质表面。使用这些技术，很明显轴膜下细胞骨架的主要成分是微丝和微管。观察到微丝通过细链的特殊网络与轴膜相连，在轴膜下方形成点状簇。这些细丝用重酶解肌球蛋白进行标记，呈现出特征性的箭头状外观。微管被观察到与轴膜平行排列，并嵌入细链的精细三维网络中。对轴突素聚集体的体外观察和免疫电子显微镜分析表明，微管周围的这种精细网络主要由轴突素组成。一些微管沿着轴膜擦过，并通过细长的链与轴膜横向相连。因此，我们得出结论，乌贼巨大轴突的轴膜因其下方的细胞骨架而特化为两个区域（微管相关区域和微丝相关区域）。
