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.
Eur J Cell Biol
; 91(5): 413-9, 2012 May.
Article
de En
| MEDLINE
| ID: mdl-22365812
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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Télophase
/
Nocodazole
/
Modulateurs de la polymérisation de la tubuline
/
Points de contrôle du cycle cellulaire
/
Composés hétérocycliques avec 4 noyaux ou plus
/
Anaphase
/
Métaphase
Limites:
Humans
Langue:
En
Journal:
Eur J Cell Biol
Année:
2012
Type de document:
Article
Pays d'affiliation:
Japon