Asai D J, Remolona N M
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Dev Biol. 1989 Apr;132(2):398-409. doi: 10.1016/0012-1606(89)90236-4.
The neuronal cells of vertebrates express two beta-tubulin isotypes, called Class II and Class III, that are neuronal specific. In order to determine the distribution of the minor Class III isotype, site-directed antibodies were raised to synthetic peptides representing the carboxyl terminal, isotype-defining domains of the tubulins. These antibodies were applied to PC12 cells at various stages of differentiation. The Class III isotype was found to be expressed in undifferentiated PC12 cells as well as in cells at every stage of differentiation. The concentration of the Class III isotype, relative to the total beta-tubulin complement, did not change significantly. Indirect double immunofluorescence microscopy demonstrated that the Class III isotype was found in the soma and the neurites of differentiated PC12 cells; this spatial pattern of Class III expression paralleled the total beta-tubulin pattern. Although the anti-Class III antiserum could stain in vitro assembled neuronal microtubules in a filamentous pattern, a close examination of the Class III staining pattern in flattened PC12 cells revealed that this isotype was not incorporated into the nonaxoplasmic array of microtubules. Rather, the Class III isotype was localized in a nonfilamentous, granular pattern that was not readily extracted with nonionic detergent. Cells treated with taxol and then flattened and stained showed that the Class III isotype could be induced to assemble into microtubule bundles by taxol. Thus, the minor neuronal beta-tubulin isotype appears to be spatially specialized in its pattern of expression.
脊椎动物的神经元细胞表达两种β-微管蛋白亚型，称为II类和III类，它们是神经元特有的。为了确定较少的III类亚型的分布，制备了针对代表微管蛋白羧基末端、亚型定义结构域的合成肽的定点抗体。这些抗体应用于不同分化阶段的PC12细胞。发现III类亚型在未分化的PC12细胞以及每个分化阶段的细胞中均有表达。相对于总的β-微管蛋白总量，III类亚型的浓度没有显著变化。间接双重免疫荧光显微镜检查表明，III类亚型存在于分化的PC12细胞的胞体和神经突中；III类亚型的这种空间表达模式与总的β-微管蛋白模式相似。尽管抗III类抗血清可以以丝状模式对体外组装的神经元微管进行染色，但仔细检查扁平PC12细胞中的III类染色模式发现，这种亚型并未整合到微管的非轴质阵列中。相反，III类亚型以非丝状、颗粒状模式定位，这种模式不易被非离子去污剂提取。用紫杉醇处理后再扁平并染色的细胞表明，紫杉醇可以诱导III类亚型组装成微管束。因此，较小的神经元β-微管蛋白亚型在其表达模式上似乎在空间上具有特异性。
