Spindle checkpoint protein dynamics at kinetochores in living cells
Spindle checkpoint protein dynamics at kinetochores in living cells
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DOI：
10.1016/j.cub.2004.05.053
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发表时间：
2004-06-08
期刊：
影响因子：
9.2
通讯作者：
Salmon, ED
中科院分区：
文献类型：
--
作者：
Howell, BJ;Moree, B;Salmon, ED
背景资料：为了测试目前的模型，以了解未连接和紧张的着丝粒如何阻止Cdc 20在整个纺锤体和细胞质中激活后期促进复合物/细胞周期体（APC/C），我们使用GFP融合和活细胞成像来量化活PtK 2细胞有丝分裂期间着丝粒处纺锤体检查点蛋白Mad 1，Mad 2，Bub 1，BubR 1，Mps 1和Cdc 20的丰度和动力学。前中期未连接的动粒平均仅结合每个纺锤体检查点蛋白的总细胞池的一小部分（估计为500-5000个分子）。光漂白后的荧光恢复（FRAP）的测量显示GFP-Cdc 20和GFP-BubR 1在未连接的动粒处表现出双相指数动力学，其中类似于50%显示非常快的动力学（t（1/2）类似于1 - 3s），并且类似于50%显示类似于GFP-Mad 2和GFP-Bub 3的单指数动力学（t（1/2）为21-23 s）的较慢动力学。GFP-Cdc 20的慢相可能代表与Mad 2的复合物形成，因为它是张力不敏感的，并且与快相不同，它不存在于缺乏Mad 2但保留Cdc 20的中期着丝粒，并且不存在于Cdc 20衍生物GFP-Cdc 20（Delta 1 -167）的未连接的前中期着丝粒，其缺乏主要的Mad 2结合结构域但保留着丝粒定位。GFP-Mps 1在未连接的动粒上表现出单指数动力学，t（1/2）接近10 s，而大多数GFP-Mad 1和GFP-Bub 1是更稳定的组分。我们的数据支持检查点激活的催化模型，其中Mad 1和Bub 1主要驻留，Mad 2不含Mad 1，BubR 1和Bub 3不含Bub 1，Cdc 20，和Mps 1动态交换的扩散等待后期信号的一部分，和Mad 2相互作用与Cdc 20在未连接的着丝粒。
Background: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells.Results: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with similar to50% displaying very fast kinetics (t(1/2) of similar to1-3 s) and similar to50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t(1/2) of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20(Delta1-167), which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t(1/2) of similar to10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components.Conclusions: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.