Bré M H, Kreis T E, Karsenti E
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
J Cell Biol. 1987 Sep;105(3):1283-96. doi: 10.1083/jcb.105.3.1283.
The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.
通过用抗中心体和抗微管蛋白抗体进行双重免疫荧光标记，在成纤维细胞（Vero）和上皮细胞（PtK2、马-达二氏犬肾[MDCK]）中分析了中心体的微管成核活性。在Vero细胞中，大多数微管从中心体发出，而MDCK细胞的微管网络似乎是非中心体成核且随机排列的。PtK2细胞中的微管组织模式介于典型成纤维细胞和上皮细胞中观察到的模式之间。在Vero和PtK2细胞中，两个中心粒圆柱体紧密相连并靠近细胞核。然而，在MDCK细胞中，它们明显分离，电子显微镜显示它们仅形成少数微管。通过用不同浓度的诺考达唑处理这些不同的细胞系，检测了中心体微管和非中心体微管的稳定性。1.6微摩尔诺考达唑诱导Vero细胞中的微管几乎完全解聚；在PtK2细胞中仍有一些由中心体形成的微管，而在MDCK细胞中许多非中心体微管抵抗了这种处理。在通过高浓度诺考达唑（33微摩尔）完全解体后释放，MDCK细胞中的中心体微管和非中心体微管以相似的动力学重新生长。在重新生长过程中，中心体微管比非中心体微管更早对1.6微摩尔诺考达唑产生抗性，尽管后者最终占主导。我们认为，在MDCK细胞中，微管如动态不稳定性模型所提出的那样生长和收缩，但存在一些因素阻止它们完全解聚。这为重新伸长创造了种子，与从中心体的成核竞争。通过使用特异性抗体，我们已经表明，MDCK细胞中对诺考达唑有抗性且数量丰富的微管子集包含去酪氨酸化的α-微管蛋白（谷氨酰胺微管蛋白）。另一方面，诺考达唑去除后最早重新生长的微管仅包含酪氨酸化微管蛋白。仅在微管重新生长30分钟后才能检测到谷氨酰胺微管蛋白。这有力地支持了以下假设：α-微管蛋白去酪氨酸化主要发生在“长寿”微管上，而不是稳定过程的原因。我们在紫杉醇处理的细胞中发现的谷氨酰胺微管蛋白量的增加也支持了这一点。
