ULTRASTRUCTURAL COLOCALIZATION OF TYROSINATED AND DETYROSINATED ALPHA-TUBULIN IN INTERPHASE AND MITOTIC CELLS
ULTRASTRUCTURAL COLOCALIZATION OF TYROSINATED AND DETYROSINATED ALPHA-TUBULIN IN INTERPHASE AND MITOTIC CELLS
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DOI：
10.1083/jcb.103.5.1883
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发表时间：
1986-11-01
影响因子：
7.8
通讯作者：
DEBRABANDER, M
中科院分区：
文献类型：
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作者：
GEUENS, G;GUNDERSEN, GG;DEBRABANDER, M
特异性肽抗体的免疫荧光先前已经确定，酪氨酸化（Tyr）和去酪氨酸化（Glu）微管蛋白是由. α的cooh末端翻译后修饰产生的两种。Gundersen, g.g.， M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789)。在本研究中使用的两种细胞类型CV1和PtK2细胞中，光镜免疫金染色观察到类似的结果；大多数微管被Tyr抗体染色，而只有少数被Glu抗体染色。我们用电子显微镜检查了免疫金染色的制剂，以扩展这些结果。总的来说，电镜定位证实了光镜水平上得到的结果：CV1和PtK2细胞中的大多数微管几乎连续地标记有Tyr抗体，而只有少数微管大量标记有Glu抗体。然而，与光镜染色相比，我们发现CV1和PtK2细胞间期和有丝分裂的所有微管在电镜水平上都含有可检测的Tyr和Glu免疫反应性。在特定细胞器（例如，线粒体或中间丝）附近的微管中没有观察到任何一种的特定定位。定量测定各间期和中期微管中Glu和Tyr免疫反应性的相对水平表明，所有类型的纺锤体微管（即着丝点、极性微管和星状微管）具有几乎相同的Glu免疫反应性；该水平的Glu免疫反应性低于所有间期微管。大多数间期微管具有低水平的Glu免疫反应性，而少数具有相对较高的水平；后者对应于形态上弯曲的微管。定量测定沿单个微管片段中Tyr和Glu免疫反应性的相对水平表明，给定微管中Tyr（或Glu）微管蛋白的水平沿其长度是均匀的。了解具有不同水平的Tyr和Glu微管蛋白的微管是如何产生的，对于理解酪氨酸化/去酪氨酸化在微管功能中的作用将是重要的。此外，两种不同水平的微管共存可能对体内微管动力学具有重要意义。
Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of .alpha.-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells; most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that the all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.