Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase
Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase
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DOI：
10.1016/j.cub.2004.09.086
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发表时间：
2004-11-09
期刊：
影响因子：
9.2
通讯作者：
Bloom, K
中科院分区：
文献类型：
--
作者：
Pearson, CG;Yeh, E;Bloom, K
凭借单微管附着，芽殖酵母着丝粒为理解与动态微管正端的协同连接以实现染色体振荡和定位提供了一个极好的系统。对着丝粒蛋白进行荧光标记表明，在中期，平均而言，所有着丝粒聚集在一起，与它们的姐妹着丝粒明显分开，并且与各自的纺锤体极等距定位。然而，着丝粒附近的单个荧光染色体标记会短暂地与其姐妹标记重新结合，并从纺锤体的一侧振荡到另一侧。为了调和平均着丝粒位置与单个着丝粒近端标记之间明显的差异，我们利用光漂白后的荧光恢复来测量组蛋白 - H3变体Cse4p/CENP - A的稳定性。新合成的Cse4p在DNA复制期间取代旧的蛋白质。一旦组装完成，Cse4 - GFP在有丝分裂期间是着丝粒的一个物理稳定成分。这使我们能够追踪每个纺锤体半区内的着丝粒动态。着丝粒稳定地附着在动态微管上，并且在纺锤体两半之间转换方向或位置的发生率较低。姐妹染色单体附着的转换可能与S期的Cse4p交换和早期着丝粒组装同时发生；这将促进染色体与每个纺锤体极的附着混合。一旦达到双向定向，着丝粒很少会超出其近端的半个纺锤体范围。
With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/ CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.