Tsukita S, Usukura J, Tsukita S, Ishikawa H
Neuroscience. 1982;7(9):2135-47. doi: 10.1016/0306-4522(82)90125-7.
The organization of the cytoskeleton in myelinated axons of the rat has been analyzed without chemical fixation in replicas of deep-etched materials after rapid freezing. Freeze-etch replicas of trigeminal nerves provided three-dimensional views of the well-developed cytoskeleton inside axons. In these preparations, the axonal cytoskeleton was seen to be composed of longitudinally-oriented microtubules and neurofilaments which were interconnected by slender strands. Such strands also connected membranous organelles with microtubules and neurofilaments. After Triton X-100 extraction, the neurofilament-associated interconnecting strands (cross-linking filaments) persisted, indicating that they are not artifactual products of soluble protein condensation during freeze-etching. In non-extracted axons many granular structures were closely associated with cytoskeletal components. These granular elements were not seen after Triton treatment. These findings, together with fluorographic analyses, suggest that the granular structures may represent. These findings, together with fluorographic analyses, suggest that the granular structures may represent slowly transported "soluble proteins' in axoplasm. This freeze-etch replica study, without any chemical fixation, substantiates the reality of the axonal cytoskeleton which is directly involved in the axonal transport. Furthermore, using this approach ultrastructural evidence was obtained of the close association of membranous structures with the cytoskeleton.
在快速冷冻后深蚀刻材料的复制品中，未进行化学固定就对大鼠有髓轴突中的细胞骨架组织进行了分析。三叉神经的冷冻蚀刻复制品提供了轴突内发育良好的细胞骨架的三维视图。在这些制备物中，可以看到轴突细胞骨架由纵向排列的微管和神经丝组成，它们通过细长的链相互连接。这些链还将膜性细胞器与微管和神经丝连接起来。用 Triton X - 100 提取后，与神经丝相关的互连链（交联丝）仍然存在，这表明它们不是冷冻蚀刻过程中可溶性蛋白质凝聚的人为产物。在未提取的轴突中，许多颗粒结构与细胞骨架成分紧密相关。Triton 处理后未见到这些颗粒成分。这些发现与放射自显影分析一起表明，这些颗粒结构可能代表轴浆中缓慢运输的“可溶性蛋白质”。这项未进行任何化学固定的冷冻蚀刻复制品研究证实了直接参与轴突运输的轴突细胞骨架的真实性。此外，通过这种方法获得了膜性结构与细胞骨架紧密关联的超微结构证据。
