Hirokawa N
J Cell Biol. 1982 Jul;94(1):129-42. doi: 10.1083/jcb.94.1.129.
The elaborate cross-connections among membranous organelles (MO), microtubules (MT), and neurofilaments (NF) were demonstrated in unifixed axons by the quick-freeze, deep-etch, and rotary-shadowing method. They were categorized into three groups: NF-associated cross-linker, MT-associated cross-bridges, and long cross-links in the subaxolemmal space. Other methods were also employed to make sure that the observed cross-connections in the unfixed axons were not a result of artifactual condensation or precipitation of soluble components or salt during deep-etching. Axolemma were permeablized either chemically (0.1% saponin) or physically (gentle homogenization), to allow egress of their soluble components from the axon; or else the axons were washed with distilled water after fixation. After physical rupture of the axolemma or saponin treatment, most of the MO remained intact. MT were stabilized by adding taxol in the incubation medium. Axons prepared by these methods contained many longitudinally oriented NF connected to each other by numerous fine cross-linkers (4-6 nm in diameter, 20-50 nm in length). Two specialized regions were apparent within the axons: one composed of fascicles of MT linked with each other by fine cross-bridges; the other was in the subaxolemmal space and consisted of actinlike filaments and a network of long cross-links (50-150 nm) which connected axolemma and actinlike filaments with NF and MT. F-actin was localized to the subaxolemmal space by the nitrobenzooxadiazol phallacidin method. MO were located mainly in these two specialized regions and were intimately associated with MT via fine short (10-20 nm in length) cross-bridges. Cross-links from NF to MO and MT were also common. All these cross-connections were observed after chemical extraction or physical rupture of the axon; however, these procedures removed granular materials which were attached to the filaments in the fresh unextracted axons. The cross-connections were also found in the axons washed with distilled water after fixation. I conclude that the cross- connections are real structures while the granular material is composed of soluble material, probably protein in nature.
通过快速冷冻、深度蚀刻和旋转阴影法，在未固定的轴突中证实了膜性细胞器（MO）、微管（MT）和神经丝（NF）之间复杂的交叉连接。它们被分为三组：NF相关交联剂、MT相关交叉桥以及轴膜下间隙中的长交联。还采用了其他方法来确保在未固定轴突中观察到的交叉连接不是深度蚀刻过程中可溶性成分或盐的人为凝聚或沉淀的结果。轴膜通过化学方法（0.1%皂角苷）或物理方法（轻柔匀浆）使其通透，以允许其可溶性成分从轴突中流出；或者在固定后用蒸馏水冲洗轴突。在轴膜物理破裂或皂角苷处理后，大多数MO保持完整。通过在孵育培养基中添加紫杉醇来稳定MT。通过这些方法制备的轴突包含许多纵向排列的NF，它们通过许多精细的交联剂（直径4 - 6纳米，长度20 - 50纳米）相互连接。轴突内有两个特殊区域很明显：一个由通过精细交叉桥相互连接的MT束组成；另一个在轴膜下间隙，由肌动蛋白样细丝和长交联网络（50 - 150纳米）组成，该网络将轴膜和肌动蛋白样细丝与NF和MT连接起来。通过硝基苯并恶二唑鬼笔环肽法将F - 肌动蛋白定位到轴膜下间隙。MO主要位于这两个特殊区域，并通过短的精细（长度10 - 20纳米）交叉桥与MT紧密相连。从NF到MO和MT的交联也很常见。在轴突化学提取或物理破裂后观察到了所有这些交叉连接；然而，这些操作去除了新鲜未提取轴突中附着在细丝上的颗粒物质。在固定后用蒸馏水冲洗的轴突中也发现了交叉连接。我的结论是，交叉连接是真实结构，而颗粒物质由可溶性物质组成，可能本质上是蛋白质。
