Mad2 and BubR1 function in a single checkpoint pathway that responds to a loss of tension
Mad2 and BubR1 function in a single checkpoint pathway that responds to a loss of tension
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DOI：
10.1091/mbc.e02-03-0137
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发表时间：
2002-10-01
影响因子：
3.3
通讯作者：
Salmon, ED
中科院分区：
文献类型：
--
作者：
Shannon, KB;Canman, JC;Salmon, ED
纺锤体检查点监测着丝点的微管附着和张力，以确保正确的染色体分离。此前，PtK1 细胞在低温条件下（23 摄氏度）显示出明显的有丝分裂延迟，尽管其着丝粒微管数量正常。在 23°C 下，我们发现 PtK1 细胞平均保持中期 101 分钟，而在 37°C 下细胞为 21 分钟。通过注射 Mad2 抑制剂消除了 23℃ 的中期延迟，表明 Mad2 和纺锤体检查点是中期延长的原因。活细胞成像显示，着丝粒 Mad2 在染色体大会后不久就无法检测到。对中期姐妹着丝粒之间拉伸的测量发现，23°C 时张力下降了 24%，并且与 37°C 相比，23°C 时中期着丝粒表现出更高水平的 3F3/2、Bub1 和 BubR1。显微注射抗 BubR1 抗体消除了 23°C 的中期延迟，表明 BubR1 较高的着丝粒水平可能导致延迟。破坏 Mad2 和 BubR1 功能可在与单一抑制相同的时间诱导后期，表明这些检查点基因在同一途径中发挥作用。我们得出的结论是，随着着丝粒微管的完整补充，着丝粒张力的降低会诱导与着丝粒 3F3/2、Bub1 和 BubR1 标记量升高相关的检查点依赖性中期延迟。
The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23degreesC) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23degreesC, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37degreesC. The metaphase delay at 23degreesC was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23degreesC, and metaphase kinetochores at 23degreesC exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37degreesC. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23degreesC, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.