Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-α tubulin
Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-α tubulin
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DOI：
10.1091/mbc.12.4.971
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发表时间：
2001-04-01
影响因子：
3.3
通讯作者：
Wadsworth, P
中科院分区：
文献类型：
--
作者：
Rusan, NM;Fagerstrom, CJ;Wadsworth, P
用绿色荧光蛋白（GFP）-α微管蛋白构建体转染LLCPK-1细胞，并建立永久表达GFP-α微管蛋白的细胞系（LLCPK-1 α）。LLCPK-1 α的有丝分裂指数和倍增时间与亲本细胞无显著差异。定量免疫印迹显示，LLCPK-1 α细胞中17%的微管蛋白是GFP-微管蛋白;未标记的微管蛋白水平降低至亲本细胞中的82%。微管动态不稳定性的参数进行了比较间期LLCPK-1 α和亲本细胞注射罗丹明标记的微管蛋白。动态不稳定性在两种情况下非常相似，表明LLCPK-1 α细胞是分析整个细胞周期微管动力学的有用工具。比较星形微管在有丝分裂中的行为与微管在间期的行为表明，与间期细胞相比，有丝分裂中灾难的频率增加了两倍，拯救的频率减少了近四倍。微管处于衰减状态或停顿状态的时间百分比也显著减少，从间期的73.5%减少到有丝分裂的11.4%。微管伸长和快速缩短的速率没有改变;有丝分裂的总体动力性增加了3.6倍。微管从中心体和一个子集的差异稳定的星形微管的释放也被观察到。这些结果提供了哺乳动物细胞有丝分裂微管动力学的第一个定量测量。
LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1 alpha). The mitotic index and doubling time for LLCPK-1 alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1 alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1 alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1 alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.