Using a live cell mitosis biosensor to probe the mechanism of the mitotic clock: From single molecule to gene family studies

The timing of mitosis is essential for high fidelity cell duplication. However, measurements of the temporal control of the mitotic clock have been challenging. Here I present a fluorescent mitosis biosensor capable of reporting mitotic durations and morphologies using wide-field fluorescence microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 minutes. The sensitivity of this assay allowed us to observe delays in mitotic timing at concentrations of taxol 100-fold below previous minimal effective doses. Inactivation of the checkpoint by targeting of 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. Mitotic timing experiments in Hela cells expressing constitutively active and dominant negative versions of the small GTPase Ran resulted in dramatic increases in the duration of prometaphase. These experimental results provide evidence to support a theoretical model that predicts a gradient of RanGTP introduces a spatial bias into microtubule dynamics that is necessary for effective 'Search and Capture' of kinetochores by microtubules during prometaphase.; The mitosis biosensor is a valuable screening tool to search for novel mitotic regulators. I used the biosensor with a library of Dicer generated small interfering RNAs (d-siRNAs) targeting 96 members of the of Ras small GTPase superfamily. The initial results from this loss of function screen produced 21 proteins that significantly altered mitotic timing. The knockdown of 18 small GTPases increased the duration of NEB to anaphase, and knockdown of three others significantly decreased this duration. Knockdown of Ran, Cdc42h and TC10 caused defects in chromosome organization, while knockdown of Rho D, Rho B and three members of the Rab subfamily Rab 11a, 25, and 27B caused late cytokinesis defects. Expression of the mutant versions of the small GTPases corroborated these results but, also clearly showed that the Rho, and Rab subfamily members are playing a major role during cytokinesis and perturbation of these signaling modules results in cytokinesis defects.