Soltys B J, Borisy G G
J Cell Biol. 1985 May;100(5):1682-9. doi: 10.1083/jcb.100.5.1682.
Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.
通过半抗原介导的免疫细胞化学方法研究了体内微管组装情况。用二氯三嗪基氨基荧光素（DTAF）对微管蛋白进行衍生化处理，然后将其显微注射到处于分裂间期的活的哺乳动物细胞中。通过双标记免疫荧光在单个微管水平确定掺入位点。用抗荧光素抗体和与荧光素偶联的二抗定位半抗原化的微管蛋白。用抗微管蛋白抗体和与罗丹明偶联的二抗鉴定总微管。与最近的研究结果（Salmon, E. D., 等人，1984年，《细胞生物学杂志》，99:2165 - 2174；Saxton, W. M., 等人，1984年，《细胞生物学杂志》，99:2175 - 2186）相反，那些研究表明微管蛋白在体内沿着微管的全长掺入，而我们发现分裂间期细胞中的微管组装在体内如同在体外一样，是一个末端介导的过程。向细胞周边辐射的微管仅在其远端，即正端掺入DTAF - 微管蛋白。我们还发现，一部分与中心体相连的微管在其整个长度上都掺入了DTAF - 微管蛋白，这表明中心体能够促使新微管的形成。
