Probing the precision of the mitotic clock with a live-cell fluorescent biosensor.
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.
Department of Molecular Pharmacology, W200 Clark, 318 Campus Drive, Stanford University Medical School, Stanford, California 94305, USA.
Joshua T
JT
Jones
Jason W
JW
Myers
James E
JE
Ferrell
Tobias
T
Meyer
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods*; D008938:Mitosis / Q000502:physiology*; D051381:Rats