Webster D R, Wehland J, Weber K, Borisy G G
Laboratory of Molecular Biology, University of Wisconsin, Madison 53706.
J Cell Biol. 1990 Jul;111(1):113-22. doi: 10.1083/jcb.111.1.113.
The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.
通过实验将主要为酪氨酸化的微管（MT）阵列转化为去酪氨酸化（Glu）阵列，然后检测MT稳定性，从而直接研究了α微管蛋白去酪氨酸化与微管（MT）稳定性之间的关系。在向小鼠瑞士3T3细胞显微注射针对酪氨酸化酶——微管蛋白酪氨酸连接酶的抗体后约1小时，MTs的Glu免疫染色荧光增强。去酪氨酸化在12小时后几乎完全完成，并持续30 - 35小时，之后才再次检测到MTs中的酪氨酸化亚基。通过先注射生物素化或异硫氰酸罗丹明标记的微管蛋白，然后分别通过半抗原介导的免疫细胞化学或光漂白后的荧光恢复来测量整体周转率，对这些经实验去酪氨酸化的MTs的稳定性进行了测试。通过这两种方法，发现周转率同样很快，半衰期约为3分钟。作为对MT稳定性的最终测试，将抗体注射细胞中乙酰化微管蛋白的染色水平与相邻未注射细胞中的染色水平以及用紫杉醇稳定MTs的细胞中观察到的染色水平进行了比较。尽管在注射抗体的细胞和紫杉醇处理的细胞中都观察到了强烈的Glu染色，但仅在紫杉醇稳定的MTs中观察到乙酰化微管蛋白染色增加，这表明MTs并未通过去酪氨酸化而稳定。总之，这些结果清楚地表明，去酪氨酸化不会直接赋予MTs稳定性。因此，在这些细胞系和其他细胞系中观察到的稳定MTs必定是通过另一种机制产生的，并且可能在其稳定后发生了翻译后修饰。
