title: 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.
abstract: 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 μM 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.
authors.affiliation: Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsui
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Mai
authors.initials: M
authors.lastname: Okamoto
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fukumoto
authors.forename: Naoto
authors.initials: N
authors.lastname: Yamaguchi
mesh_terms: D000705:Anaphase / Q000187:drug effects* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D059447:Cell Cycle Checkpoints / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D006801:Humans; D008677:Metaphase / Q000187:drug effects* / Q000502:physiology; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology*; D013692:Telophase / Q000187:drug effects* / Q000502:physiology; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D006576:Heterocyclic Compounds, 4 or More Rings; D050257:Tubulin Modulators; C472645:blebbistatin; D024462:Myosin Type II; D015739:Nocodazole