Hirokawa N, Bloom G S, Vallee R B
J Cell Biol. 1985 Jul;101(1):227-39. doi: 10.1083/jcb.101.1.227.
Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.
用神经毒性剂β，β'-亚氨基二丙腈（IDPN）处理过的大鼠轴突，通过快速冷冻、深度蚀刻电子显微镜进行了检查。微管在轴突的中央区域形成束状，而神经丝则被分隔到外周。大多数膜结合细胞器，大概包括那些参与快速轴突运输的细胞器，都与微管区域相关。快速冷冻、深度蚀刻电子显微镜提供的高分辨率显示，微管被一个广泛的细丝网络所覆盖，这些细丝既用于交联微管，又用于将它们与膜结合细胞器相互连接。细丝上装饰有颗粒物质，尺寸不规则。它们在新鲜轴突中单独出现或作为一个广泛的吻合网络出现。在新鲜的、经皂角苷提取的或醛固定的组织中都观察到了与微管相关的细丝。为了进一步探究与微管相关细丝的特性，从脑组织中纯化并含有高分子量微管相关蛋白MAP 1和MAP 2的微管，通过快速冷冻、深度蚀刻电子显微镜进行了检查。纯化的微管通过一个细丝网络连接，其外观与在IDPN处理的轴突中观察到的非常相似。仅由微管蛋白组成且缺乏MAPs的对照微管制剂没有相关细丝。为了了解轴突微管束中存在哪些MAPs，使用针对MAP 1A、MAP 1B、MAP 2和微管蛋白的抗体，通过免疫荧光显微镜检查了IDPN处理大鼠的轴突切片。在IDPN处理的轴突中，抗MAP 2染色仅能勉强检测到，这与早期观察结果一致。抗MAP 1A和抗MAP 1B能使IDPN处理的轴突强烈染色，且染色仅限于微管区域。此外，使用胶体金标记二抗的超薄切片免疫电子显微镜显示，抗MAP 1A和抗MAP 1B都能使微管之间的模糊丝状结构染色。鉴于早期的研究表明快速运输与IDPN处理的轴突中的微管区域相关，现在看来MAP 1A和MAP 1B可能在这个过程中起作用。我们认为MAP 1A和MAP 1B是轴突中与微管相关的纤维状基质的主要成分。
