Fig. 4. Reduced mitotic checkpoint activity in ncp cells. Histograms from flow cytometric analysis of DAPI-stained cells from 24-hour postfertilization wild-type and ncp embryos. (Left) FACs profile of cells collected from embryos (raised at 25°C) after a 4-hour incubation at 33°C. The majority of cells in wild-type and ncp samples had a 2N nuclear content. (Right) FACs profile of cells collected from embryos treated with 1 µg/ml nocodazole during the 4-hour 33°C incubation. A large 4N peak, representing cells that have arrested in mitosis, appeared in both wild-type and ncp histograms. However, note the substantial >4N subpopulation in the ncp histogram (arrows). Such cells presumably reflect those that failed to arrest in mitosis and continued to synthesize DNA despite the absence of mitotic spindles. High DNA content (>2% of cells with DNA content over 4N) was not observed in cell suspensions from nocodazole-treated wild-type embryos (n=10), but was seen in 10 of 12 suspensions from treated ncp embryos. Thus, mitotic checkpoint activity is reduced in ncp embryonic cells, indicating that the I843K mutation disrupts Mps1 function in zebrafish.

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