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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex
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Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

机译：β-微管蛋白抗原决定簇在嗜热四膜菌和尾草履膜皮层中的独特定位

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摘要

Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against beta-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against gamma- tubulin, detyrosinated alpha-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta 81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins.
机译：纤毛中许多高度组织化的微管排列位于含有膜囊泡和液泡的皮质区域。在嗜热四膜菌和尾草履虫中，用针对β-微管蛋白的单克隆抗体TU-06进行的免疫荧光显微镜检查仅显示了该皮质区域的明显染色，而纤毛和其他微管结构未染色。通过免疫印迹和通过用纯化的微管蛋白预吸收抗体来确认抗体的特异性。用针对γ-微管蛋白，脱酪氨酸的α-微管蛋白和centrin的抗体进行的双标记免疫荧光显示，TU-06表位位于基底体区域之外。薄切片的免疫金电子显微镜也证实了这一点。猪脑β-微管蛋白的蛋白水解消化与固定的重叠肽段的肽扫描相结合，揭示了该表位在β-微管蛋白区域β81-95中，该区域在系统发育上高度保守。由于已知β-微管蛋白的翻译后修饰位于该区域之外，因此观察到的染色模式不能解释为修饰微管蛋白的亚细胞螯合的证据。表位的有限分布可能反而反映了TU-06表位暴露对微管排列中微管蛋白分子构象的依赖性或对相互作用蛋白的差异掩盖。

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