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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We would like to thank Guowei Fang, Thierry Galvez, Marc Fivaz and Angie Hahn for critical reading of the manuscript. We would also like to thank G. Fang for his kind gift of the Mad2 antibody, Tom Wehrman for help with creating the stable line and the Meyer and Ferrell labs for their support. This work was supported by the National Institutes of Health.
The authors declare no competing financial interests.
Location of the biosensor during mitosis. Asynchronous RBL-FMB cells were imaged every 15 minutes with a CFP/YFP spinning disc confocal system. 3 cells that enter mitosis are identified by the white arrows. The scale bar in the lower left corner represents 20 µm and the time is in hours:minutes in the lower right corner. (MOV 204 kb)
Localization of the biosensor as monitored by total internal reflection fluorescence (TIRF) microscopy. RBL-FMB cells were imaged with a prism based CFP/YFP TIRF system every two minutes. The rectangular region highlights one cell undergoing mitosis with six cells in total going through mitosis during the course of the time lapse. The scale bar in the lower left corner represents 20 µm and the time is in hours:minutes in the lower right corner. (MOV 1890 kb)
A 10X TIRF view of cycling RBL-FMB cells. RBL-FMB cells were imaged with a prism based CFP/YFP TIRF system. 70 cells undergo mitosis in the field of view during this 5 hour experiment. 75 frames of 149 are represented here. The scale bar in the lower left corner represents 50 µm and the time is represented in hours:minutes in the lower right corner. (MOV 6490 kb)
40X TIRF and transmitted light time lapse of cycling mitotic cells. RBL-FMB cells were imaged with a prism based CFP/YFP TIRF system with near simultaneous transmitted light exposures. The scale bar in the lower left corner represents ten mm and the time is represented in hours:minutes in the lower right corner. (MOV 1325 kb)
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Jones, J., Myers, J., Ferrell, J. et al. Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nat Biotechnol 22, 306–312 (2004) doi:10.1038/nbt941
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