Rieder C L, Cole R W, Khodjakov A, Sluder G
Division of Molecular Medicine, Wadsworth Center, Albany, New York 12201-0509, USA.
J Cell Biol. 1995 Aug;130(4):941-8. doi: 10.1083/jcb.130.4.941.
During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.
在Ptk1细胞有丝分裂期间，直到最后一条单极定向染色体与纺锤体形成双极附着后平均23±1分钟，后期才开始——这一过程可能需要3小时（Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301 - 1310）。为了确定这种细胞周期检查点信号的性质及其产生位点，我们通过视频显微镜观察PtK1细胞，在通过激光照射破坏特定染色体区域之前和之后进行观察。在最后一条单极定向染色体的着丝粒（及其相关的两个姐妹动粒）被破坏后17±1分钟，检查点被解除，细胞进入后期。因此，检查点机制监测单极定向染色体着丝粒中产生的后期抑制因子。接下来，在存在一条单极定向染色体的情况下，我们破坏一条双极定向染色体上的一个动粒，以产生第二条没有未附着动粒的单极定向染色体。在这种条件下，在未照射的单极定向染色体双极定向后24±1.5分钟，实验产生的单极定向染色体存在时后期开始。这一结果表明，检查点信号不是由单极定向染色体上附着的动粒产生的，也不是在整个着丝粒区域产生的。最后，我们选择性地破坏最后一条单极定向染色体上未附着的动粒。在这种条件下，操作后20±2.5分钟细胞进入后期，未使受照射的染色体排列到赤道板。对这些处于后期的细胞进行相关的光学显微镜/电子显微镜观察，证实未附着染色单体上没有动粒。总之，我们的数据表明，单极定向的PtK1染色体未附着动粒中或其附近的分子抑制中期 - 后期转换。
