Pepper D A, Brinkley B R
J Cell Biol. 1979 Aug;82(2):585-91. doi: 10.1083/jcb.82.2.585.
A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.
为了确定微管蛋白抗体是否能阻断外源性微管蛋白在动粒和中心体处的成核作用，我们开发了一种细胞裂解系统。用秋水仙酰胺预处理有丝分裂的PtK2细胞以去除所有内源性微管，然后在PIPES-EGTA-Mg++缓冲液中用Triton X-100裂解细胞。通过对薄切片细胞的电子显微镜观察确定，该过程使中心体、染色体和动粒保持完整。在1 mM GTP存在的情况下，将裂解后的细胞在37℃下暴露于磷酸纤维素纯化的微管蛋白二聚体中，导致微管在中心体和动粒处进行位点特异性成核。在随后暴露于外源性微管蛋白之前，用微管蛋白抗体处理裂解细胞制剂，几乎完全阻断了微管成核，尤其是在动粒处。用对照抗体或不含抗体的缓冲液预处理裂解细胞制剂，对中心体和动粒启动微管组装的能力没有影响。讨论了这些结果对中心体和动粒分子组成的意义。
