Synchronization and Desynchronization of Cells by Interventions on the Spindle Assembly Checkpoint.
Cell cycle checkpoints are surveillance mechanisms that sequentially and continuously monitor cell cycle progression thereby contributing to the preservation of genetic stability. Among them, the spindle assembly checkpoint (SAC) prevents the occurrence of abnormal divisions by halting the metaphase to anaphase transition following the detection of erroneous microtubules-kinetochore attachment(s). Most synchronization strategies are based on the activation of cell cycle checkpoints to enrich the population of cells in a specific phase of the cell cycle. Here, we develop a two-step protocol of sequential cell synchronization and desynchronization employing antimitotic SAC-inducing agents (i.e., nocodazole or paclitaxel) in combination with the depletion of the SAC kinase MPS1. We describe cytofluorometric and time-lapse videomicroscopy methods to detect cell cycle progression, including the assessment of cell cycle distribution, quantification of mitotic cell fraction, and analysis of single cell fate profile of living cells. We applied these methods to validate the synchronization-desynchronization protocol and to qualitatively and quantitatively determine the impact of SAC inactivation on the activity of antimitotic agents.
Department of Cardiology, Vascular Medicine and Physiology, University of Tuebingen, Tuebingen, Germany.
Mohamed
M
Jemaà
Regina Elena National Cancer Institute, Rome, Italy.
Gwenola
G
Manic
Regina Elena National Cancer Institute, Rome, Italy. iliovit@gmail.com.|Department of Biology, University of Rome "Tor Vergata", Rome, Italy. iliovit@gmail.com.
Ilio
I
Vitale
D000705:Anaphase / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints / Q000235:genetics / Q000502:physiology*; D018715:Microscopy, Video; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011123:Polyploidy; D034741:RNA, Small Interfering / Q000235:genetics; D012984:Software; D057891:Tetraploidy
D006657:Histones; D034741:RNA, Small Interfering
APC/C; Anaphase; Cell cycle; Chromosome; Microtubules; Mitosis; Polyploidy; Tetraploidy