Rodionov V, Nadezhdina E, Borisy G
Laboratory of Molecular Biology, R. M. Bock Laboratories, University of Wisconsin, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):115-20. doi: 10.1073/pnas.96.1.115.
In many animal cells, minus ends of microtubules (MTs) are thought to be capped by the centrosome whereas plus ends are free and display dynamic instability. We tested the role of the centrosome by examining MT behavior in cytoplasts from which the centrosome was removed. Cells were injected with Cy3-tubulin to fluorescently label MTs and were enucleated by using a centrifugation procedure. Enucleation resulted in a mixture of cytoplasts containing or lacking the centrosome. Fibroblast (CHO-K1) and epithelial (BSC-1) cells were investigated. In fibroblast cytoplasts containing the centrosome, MTs showed dynamic instability indistinguishable from that in intact cells. In contrast, in cytoplasts lacking the centrosome, MTs treadmilled-shortened at the minus end at about 12 micrometers/min while growing at the plus end at the same rate. The change in behavior of the plus end from dynamic instability to persistent growth correlated with an elevated level of free tubulin subunits (78% in centrosome-free cytoplasts vs. 44% in intact cells) generated by minus-end depolymerization. In contrast to fibroblast cells, in centrosome-free cytoplasts prepared from epithelial cells, MTs displayed dynamic instability at plus ends and relative stability at minus ends presumably because of specific minus-end stability factors distributed throughout the cytoplasm. We suggest that, in fibroblast cells, a minus-end depolymerization mechanism functions to eliminate errors in MT organization and that dynamic instability of MT plus ends is a result of capping of minus ends by the centrosome.
在许多动物细胞中，微管（MTs）的负极被认为由中心体加帽，而正极是自由的，并表现出动态不稳定性。我们通过检查去除中心体的胞质体中的MT行为来测试中心体的作用。向细胞中注射Cy3-微管蛋白以荧光标记MTs，并使用离心程序去核。去核导致含有或缺乏中心体的胞质体混合物。研究了成纤维细胞（CHO-K1）和上皮细胞（BSC-1）。在含有中心体的成纤维细胞胞质体中，MTs表现出与完整细胞中难以区分的动态不稳定性。相比之下，在缺乏中心体的胞质体中，MTs以约12微米/分钟的速度在负极处踏车式缩短，同时在正极处以相同速度生长。正极行为从动态不稳定性到持续生长的变化与负极解聚产生的游离微管蛋白亚基水平升高相关（无中心体的胞质体中为78%，而完整细胞中为44%）。与成纤维细胞不同，在上皮细胞制备的无中心体胞质体中，MTs在正极处表现出动态不稳定性，在负极处表现出相对稳定性，这可能是由于特定的负极稳定因子分布在整个细胞质中。我们认为，在成纤维细胞中，负极解聚机制起到消除MT组织错误的作用，MT正极的动态不稳定性是中心体对负极加帽的结果。
