Bré M H, Pepperkok R, Hill A M, Levilliers N, Ansorge W, Stelzer E H, Karsenti E
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
J Cell Biol. 1990 Dec;111(6 Pt 2):3013-21. doi: 10.1083/jcb.111.6.3013.
MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bré, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.
MDCK细胞在组织培养中达到汇合时会形成极化上皮。我们之前已经表明，伴随着细胞间连接的建立，中心粒分离，微管失去其放射状组织（巴卡略，R.，C. 安东尼，C. 多蒂，E. 卡尔森蒂，E.H.K. 施特尔泽，以及K. 西蒙斯。1989年。《细胞生物学杂志》109:2817 - 2832。布恩迪亚，B.，M.H. 布雷，G. 格里菲思，以及E. 卡尔森蒂。1990年。110:1123 - 1136）。在这项工作中，我们研究了细胞间接触建立前后微管成核的模式。我们分析了单个细胞和汇合细胞中微管的伸长率和稳定性。这是通过微注射草履虫轴丝微管蛋白并使用一种专门针对草履虫轴丝微管蛋白的抗体检测新掺入的亚基来实现的。利用先进的双免疫荧光共聚焦显微镜使得确定新成核微管的定位成为可能。我们已经表明，在单个细胞中，新成核的微管起源于集中在靠近细胞核区域的几个位点，而不是来自可能对应于一对中心粒的单个点。在汇合细胞中，新成核的微管仍然更加分散。单个微管在单个细胞和汇合细胞中的伸长率没有差异（4微米/分钟）。然而，在汇合细胞中，长寿微管的数量显著增加。在单个或亚汇合细胞中，大多数微管的半衰期小于30分钟，而在汇合单层细胞中，大量微管的半衰期大于2小时。这些结果与上述先前的观察结果一起表明，在MDCK细胞极性建立过程中，微管重组涉及微管成核活性的重新定位和微管稳定性的增加。
