Nature Biotechnology22, 306 - 312 (2004)
Published online: 8 February 2004; | doi:10.1038/nbt941
Probing the precision of the mitotic clock with a live-cell fluorescent biosensor
Joshua T Jones1, Jason W Myers1, James E Ferrell Jr2
& Tobias Meyer1
1
Department of Molecular Pharmacology, W200 Clark, 318 Campus Drive, Stanford University Medical School, Stanford, California 94305, USA.
2
Department of Molecular Pharmacology & Department of Biochemistry, 269 Campus Drive, Stanford University Medical School, Stanford, California 94305, USA.
Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.
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6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.
