Baas P W, Ahmad F J
Department of Anatomy, University of Wisconsin Medical School, Madison 53706.
J Cell Biol. 1992 Mar;116(5):1231-41. doi: 10.1083/jcb.116.5.1231.
Microtubules (MTs) in the axon have a uniform polarity orientation that is recapitulated during recovery from episodes of MT depolymerization (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu. 1984. J. Cell Biol. 99:1289-1295). This tight regulation of their organization indicates that axonal MTs are spatially regulated by discrete nucleating structures comparable in function to the centrosome. Several authors have proposed that an especially stable class of MTs in the axon may serve as these nucleating structures. In a previous report (Baas, P. W., and M. M. Black. 1990. J. Cell Biol. 111:495-509), we determined that the axons of cultured sympathetic neurons contain two classes of MT polymer, stable and labile, that differ in their sensitivity to nocodazole by roughly 35-fold. The stable and labile polymer represent long-lived and recently assembled polymer, respectively. We also determined that these two classes of polymer can be visually distinguished at the immunoelectron microscopic level based on their content of tyrosinated alpha-tubulin: the labile polymer stains densely, while the stable polymer does not stain. In the present study, we have taken advantage of these observations to directly identify MT nucleating structures in the axon. Neuron cultures were treated with nocodazole for 6 h to completely depolymerize the labile polymer in the axon, and substantially shorten the stable polymer. The cultures were then rinsed free of the drug, permitted to reassemble polymer for various periods of time, and prepared for immunoelectron microscopic localization of tyrosinated alpha-tubulin. Serial reconstruction of consecutive thin sections was undertaken to determine the spatial relationship between the stable MTs and the newly assembled polymer. All of the new polymer assembled in direct continuity with the plus ends of stable MTs, indicating that these ends are assembly competent, and hence capable of acting as nucleating structures. Our results further indicate that no self-assembly of MTs occurs in the axon, nor do any MT nucleating structures exist in the axon other than the plus ends of stable MTs. Thus the plus ends of stable MTs are the exclusive nucleating structures for MTs in the axon.
轴突中的微管（MTs）具有统一的极性方向，这种极性方向在微管解聚后的恢复过程中得以重现（海德曼，S. R.，M. A. 汉伯格，S. J. 托马斯，B. 宋，S. 林德利，以及D. 朱。1984年。《细胞生物学杂志》99:1289 - 1295）。它们这种紧密的组织调控表明，轴突微管在空间上受到与中心体功能相当的离散成核结构的调控。几位作者提出，轴突中一类特别稳定的微管可能充当这些成核结构。在之前的一份报告中（巴斯，P. W.，以及M. M. 布莱克。1990年。《细胞生物学杂志》111:495 - 509），我们确定培养的交感神经元轴突含有两类微管聚合物，即稳定型和不稳定型，它们对诺考达唑的敏感性相差约35倍。稳定型和不稳定型聚合物分别代表寿命长的和最近组装的聚合物。我们还确定，基于它们的酪氨酸化α - 微管蛋白含量，这两类聚合物在免疫电子显微镜水平上可以通过视觉区分：不稳定型聚合物染色浓密，而稳定型聚合物不染色。在本研究中，我们利用这些观察结果直接鉴定轴突中的微管成核结构。将神经元培养物用诺考达唑处理6小时，以使轴突中的不稳定型聚合物完全解聚，并显著缩短稳定型聚合物。然后将培养物冲洗掉药物，让其重新组装聚合物不同时间，接着制备用于酪氨酸化α - 微管蛋白免疫电子显微镜定位的样本。对连续的薄切片进行系列重建，以确定稳定型微管与新组装的聚合物之间的空间关系。所有新组装的聚合物都与稳定型微管的正端直接连续组装，这表明这些正端具有组装能力，因此能够充当核结构。我们的结果进一步表明，轴突中不存在微管的自组装现象，除了稳定型微管的正端外，轴突中也不存在任何微管成核结构。因此，稳定型微管的正端是轴突中微管唯一的成核结构。
