Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.
To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
Laboratory of Cell Regulation, Wadsworth Center, Albany, New York 12201-0509.
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Rieder
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Schultz
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Cole
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Sluder
D000705:Anaphase / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000502:physiology* / Q000648:ultrastructure; D003703:Demecolcine / Q000494:pharmacology; D018386:Kinetochores / Q000378:metabolism*; D007614:Macropodidae; D008677:Metaphase / Q000502:physiology; D018715:Microscopy, Video; D009685:Nuclear Envelope / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*
D017239:Paclitaxel; D003703:Demecolcine