Probing the precision of the mitotic clock with a live-cell fluorescent biosensor.

摘要: Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements the mitotic clock have been challenging. Here we present a fluorescent biosensor that monitors time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds events per experiment, found unsynchronized rat basophilic leukemia cells has marked precision with 80% completing in 32 ± 6 min. This assay further allowed us observe delays at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why clinically active low concentrations. Inactivation spindle checkpoint by targeting regulator Mad2 RNAi consistently shortened mitosis, providing direct evidence mechanism much faster lack checkpoint.