Arregui C, Busciglio J, Caceres A, Barra H S
Centro de Investigaciones en Química Biologica de Córdoba, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
J Neurosci Res. 1991 Feb;28(2):171-81. doi: 10.1002/jnr.490280204.
We have used the monoclonal antibody YL 1/2 (Tyr) specific for tyrosinated tubulin, and a polyclonal antibody (Glu) specific for detyrosinated tubulin to visualize the distribution of microtubules and microtubule assembly sites during axonal outgrowth. Cerebellar macroneurons growing in culture initially extend several short and thin neurites which have the potential to differentiate either as axons or dendrites (Ferreira and Caceres: Developmental Brain Research 49:205-213, 1989). At the onset of axonal outgrowth the Tyr antibody labels the minor neurites, the axon, and its growth cone, while the Glu antibody only shows immunoreactivity in the axonal shaft. After nocodazole treatment, the Tyr staining disappears, whereas that produced by the Glu antibody remains practically unchanged. When nocodazole was removed, tyrosinated microtubules reappeared first at the tip of the axon, in a more distal region than that occupied by detyrosinated microtubules; another focal site of tyrosinated tubulin incorporation was detected in the cell body. Incorporation of tyrosinated tubulin into growing axons was also studied after taxol treatment. After long incubation periods in the presence of taxol, the Tyr staining disappeared from the axon but remained in the cell body; however, immunoreactivity in this site was negative when the cells were preincubated in the presence of protein synthesis inhibitors. Release from taxol results in the reappearance of Tyr immunoreactivity at the distal end of the axon. Taken collectively, the present results indicate 1) that in cerebellar macroneurons axonal differentiation is accompanied by a temporal and spatial differentiation of microtubules and 2) that there is an active site of tyrosinated tubulin assembly at the tip of axonal processes, and they suggest that the highly tyrosinated domain in this region is a consequence of rapid microtubule turnover and tubulin tyrosine ligase activity.
我们使用了针对酪氨酸化微管蛋白的单克隆抗体YL 1/2（Tyr），以及针对去酪氨酸化微管蛋白的多克隆抗体（Glu），以观察轴突生长过程中微管的分布和微管组装位点。培养中的小脑大神经元最初会伸出几条短而细的神经突，这些神经突有可能分化为轴突或树突（费雷拉和卡塞雷斯：《发育脑研究》49:205 - 213，1989）。在轴突开始生长时，Tyr抗体标记较小的神经突、轴突及其生长锥，而Glu抗体仅在轴突干中显示免疫反应性。用诺考达唑处理后，Tyr染色消失，而Glu抗体产生的染色基本不变。去除诺考达唑后，酪氨酸化微管首先出现在轴突顶端，比去酪氨酸化微管占据的区域更靠远端；在细胞体中还检测到另一个酪氨酸化微管蛋白掺入的焦点位点。在用紫杉醇处理后，也研究了酪氨酸化微管蛋白掺入生长轴突的情况。在紫杉醇存在下长时间孵育后，Tyr染色从轴突消失，但仍保留在细胞体中；然而，当细胞在蛋白质合成抑制剂存在下预孵育时，该位点的免疫反应性为阴性。从紫杉醇中释放导致Tyr免疫反应性在轴突远端重新出现。综合来看，目前的结果表明：1）在小脑大神经元中，轴突分化伴随着微管的时空分化；2）在轴突末端存在一个活跃的酪氨酸化微管蛋白组装位点，并且表明该区域高度酪氨酸化的结构域是微管快速周转和微管蛋白酪氨酸连接酶活性的结果。
