Individual microtubules in the axon consist of domains that differ in both composition and stability.
Individual microtubules in the axon consist of domains that differ in both composition and stability.
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DOI：
10.1083/jcb.111.2.495
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发表时间：
1990-08
影响因子：
7.8
通讯作者：
Black, M M
中科院分区：
文献类型：
--
作者：
Baas, P W;Black, M M
我们探讨了培养的交感神经元轴突中单个微管（MT）的组成和稳定性。使用形态测定方法来定量在2微克/ml诺考达唑中不同时间后保留在轴突中的MT质量，我们观察到轴突中约48%的MT质量是不稳定的，解聚的t1/2约为5分钟，而剩余的52%的MT质量是稳定的，解聚的t1/2约为240分钟。免疫荧光分析显示轴突中的不稳定MT富含酪氨酸化的α-微管蛋白，而稳定的MT含有很少或不含酪氨酸化的α-微管蛋白，而是富含脱酪氨酸和乙酰化后的α-微管蛋白。这些结果得到了证实，定量的免疫电子显微镜分析之间的轴突MT的酪氨酸化α-微管蛋白的分布。单个MT图谱通常沿其长度沿着均匀标记酪氨酸化α-微管蛋白，或完全未标记。在诺考达唑处理的15分钟内，大约48%的MT质量被酪氨酸化，大约52%被脱酪氨酸化，并且大约85%的酪氨酸化MT被耗尽。因此，酪氨酸化或去酪氨酸化的MT谱的比例分别精确地对应于不稳定或稳定的MT的比例。我们还观察到MT图谱，其一端密集标记酪氨酸化α-微管蛋白，但另一端完全未标记。在所有这些后一种情况下，酪氨酸化的，因此不稳定的结构域，位于正端的MT，而脱酪氨酸，因此稳定的结构域位于负端的MT，并在每种情况下，有一个突然的过渡之间的两个域。基于观察到的这些MT配置文件的频率，我们估计轴突中至少有40%的MT是复合的，由稳定的脱酪氨酸结构域与不稳定的酪氨酸结构域直接连续组成。不稳定区域的极端药物敏感性表明它们是非常动态的，在轴突内迅速翻转。不稳定结构域和稳定结构域之间的直接连续性表明不稳定MT直接从稳定MT组装。我们建议，稳定的MT作为MT成核结构，在空间上调节轴突中的MT动力学。
We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining in axons after various times in 2 micrograms/ml nocodazole, we observed that approximately 48% of the MT mass in the axon is labile, depolymerizing with a t1/2 of approximately 5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a t1/2 of approximately 240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated alpha-tubulin, whereas the stable MTs contain little or no tyrosinated alpha-tubulin and are instead rich in posttranslationally detyrosinated and acetylated alpha-tubulin. These results were confirmed quantitatively by immunoelectron microscopic analyses of the distribution of tyrosinated alpha-tubulin among axonal MTs. Individual MT profiles were typically either uniformly labeled for tyrosinated alpha-tubulin all along their length, or were completely unlabeled. Roughly 48% of the MT mass was tyrosinated, approximately 52% was detyrosinated, and approximately 85% of the tyrosinated MTs were depleted within 15 min of nocodazole treatment. Thus, the proportion of MT profiles that were either tyrosinated or detyrosinated corresponded precisely with the proportion of MTs that were either labile or stable respectively. We also observed MT profiles that were densely labeled for tyrosinated alpha-tubulin at one end but completely unlabeled at the other end. In all of these latter cases, the tyrosinated, and therefore labile domain, was situated at the plus end of the MT, whereas the detyrosinated, and therefore stable domain was situated at the minus end of the MT, and in each case there was an abrupt transition between the two domains. Based on the frequency with which these latter MT profiles were observed, we estimate that minimally 40% of the MTs in the axon are composite, consisting of a stable detyrosinated domain in direct continuity with a labile tyrosinated domain. The extreme drug sensitivity of the labile domains suggests that they are very dynamic, turning over rapidly within the axon. The direct continuity between the labile and stable domains indicates that labile MTs assemble directly from stable MTs. We propose that stable MTs act as MT nucleating structures that spatially regulate MT dynamics in the axon.