Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: A novel method suitable for examining dynamic changes in proteins during mitotic progression
Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: A novel method suitable for examining dynamic changes in proteins during mitotic progression
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DOI：
10.1016/j.ejcb.2011.12.008
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发表时间：
2012-05-01
影响因子：
6.6
通讯作者：
Yamaguchi, Naoto
中科院分区：
文献类型：
--
作者：
Matsui, Yuki;Nakayama, Yuji;Yamaguchi, Naoto
有丝分裂是一个连续的过程，通过前期，中期，后期和末期将复制的染色体分离成两个子细胞。虽然已经建立了许多方法来同步细胞在细胞周期的不同阶段，这是很难同步细胞在特定的阶段，后期和末期，在有丝分裂，因为后期的持续时间短。在这里，我们表明，HeLa S3细胞在后期和末期成功地丰富了低浓度的微管解聚剂诺考达唑和肌球蛋白II抑制剂blebbistatin的组合治疗。在G1/S期从胸苷阻断释放9小时后，加入20 ng/ml但不是40 ng/ml的诺考达唑确保从诺考达唑阻滞中快速释放。随后，将细胞在50 μ M blebbistatin存在下培养20和50分钟，以分别在后期和末期富集细胞。蛋白质印迹分析证实磷酸化组蛋白H3-Ser10、磷酸化极光A/B/C和细胞周期蛋白B1在M期进展过程中下调。此外，我们展示了Src家族激酶c-Yes和c-Src的电泳迁移率变化在有丝分裂的每个阶段是如何变化的。这些结果提供了一个有用的同步方法，生化检查蛋白质动力学在M期进展。(C)2012 Elsevier GmbH. All rights reserved.
Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50 p,M blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression. (C) 2012 Elsevier GmbH. All rights reserved.