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.

[1]  D. E. Wolf,et al.  Feedback control of the metaphase-anaphase transition in sea urchin zygotes: role of maloriented chromosomes , 1994, The Journal of cell biology.

[2]  W. Earnshaw,et al.  CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase , 1994, The Journal of cell biology.

[3]  E D Salmon,et al.  Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle , 1994, The Journal of cell biology.

[4]  C. Rieder,et al.  The Events and Regulation of Anaphase Onset , 1993 .

[5]  M. Jordan,et al.  Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Ricketts,et al.  Differential expression of a phosphoepitope at the kinetochores of moving chromosomes , 1993, The Journal of cell biology.

[7]  E D Salmon,et al.  Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism , 1993, The Journal of cell biology.

[8]  M. Jordan,et al.  Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes. , 1993, Journal of cell science.

[9]  A. Murray,et al.  Creative blocks: cell-cycle checkpoints and feedback controls , 1992, Nature.

[10]  F. Spencer,et al.  Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Jordan,et al.  Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. , 1992, Journal of cell science.

[12]  T. Hunt,et al.  The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo , 1992, The Journal of cell biology.

[13]  C. Rieder,et al.  Chromosome mal-orientation and reorientation during mitosis. , 1992, Cell motility and the cytoskeleton.

[14]  Andrew W. Murray,et al.  Feedback control of mitosis in budding yeast , 1991, Cell.

[15]  B. Roberts,et al.  S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function , 1991, Cell.

[16]  M. Jordan,et al.  Mechanism of inhibition of cell proliferation by Vinca alkaloids. , 1991, Cancer research.

[17]  C. Rieder Mitosis: towards a molecular understanding of chromosome behavior. , 1991, Current opinion in cell biology.

[18]  W. Earnshaw,et al.  Role of the centromere/kinetochore in cell cycle control. , 1991, Cold Spring Harbor symposia on quantitative biology.

[19]  J. McIntosh Structural and mechanical control of mitotic progression. , 1991, Cold Spring Harbor symposia on quantitative biology.

[20]  R. Schimke,et al.  Cell line-specific differences in the control of cell cycle progression in the absence of mitosis. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. Borisy,et al.  Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis , 1990, The Journal of cell biology.

[22]  C. Rieder Formation of the astral mitotic spindle: ultrastructural basis for the centrosome-kinetochore interaction. , 1990, Electron microscopy reviews.

[23]  L. Hartwell,et al.  Checkpoints: controls that ensure the order of cell cycle events. , 1989, Science.

[24]  G. Borisy,et al.  Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly. , 1989, Journal of cell science.

[25]  Rieder Cl,et al.  The attachment of chromosomes to the mitotic spindle and the production of aneuploidy in newt lung cells. , 1989 .

[26]  C. Rieder,et al.  The attachment of chromosomes to the mitotic spindle and the production of aneuploidy in newt lung cells. , 1989, Progress in clinical and biological research.

[27]  G. Sluder Control mechanisms of mitosis--The role of spindle microtubules in the timing of mitotic events (Advances in Cell Division Research) , 1988 .

[28]  Nicklas Rb Chance encounters and precision in mitosis. , 1988 .

[29]  R. Nicklas Chance encounters and precision in mitosis. , 1988, Journal of cell science.

[30]  R. Nicklas,et al.  The forces that move chromosomes in mitosis. , 1988, Annual review of biophysics and biophysical chemistry.

[31]  C. Rieder,et al.  Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption. , 1987, Cell motility and the cytoskeleton.

[32]  G. Sluder,et al.  Control mechanisms of the cell cycle: role of the spatial arrangement of spindle components in the timing of mitotic events , 1983, The Journal of cell biology.

[33]  J. Mullins,et al.  Loss of mitotic centrosomal microtubule initiation capacity at the metaphase-anaphase transition. , 1982, European journal of cell biology.

[34]  G. Sluder Role of spindle microtubules in the control of cell cycle timing , 1979, The Journal of cell biology.

[35]  R. E. Zirkle Ultraviolet-microbeam irradiation of newt-cell cytoplasm: spindle destruction, false anaphase, and delay of true anaphase. , 1970, Radiation research.

[36]  G. Rose A separable and multipurpose tissue culture chamber. , 1954, Texas reports on biology and medicine.