Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET.
Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET.
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DOI：
10.1016/j.cub.2014.05.014
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发表时间：
2014-07-07
期刊：
影响因子：
--
通讯作者：
Joglekar AP
中科院分区：
文献类型：
--
作者：
Aravamudhan P;Felzer-Kim I;Gurunathan K;Joglekar AP
着丝粒是一个多蛋白质机器，它将染色体运动与微管(MT)聚合和解聚联系在一起。它使用至少三种MT结合蛋白的大量拷贝来产生双向运动。这些蛋白质在动粒内的纳米尺度组织在形成驱动动粒持续双向运动的机制方面起着重要作用。我们使用Förster Resonance Energy Transfer(FRET)在融合到动粒亚基上的基因编码的荧光蛋白之间重建萌芽酵母动粒的纳米级组织。我们进行了>；60 FRET和高分辨率共定位测量，涉及到必要的MT结合动粒成分：Ndc80、DAM1、Spc105和Stu2。这些测量结果表明，动粒内相邻的Ndc80复合体沿着MT的长度分布得很窄。DAM1复杂分子集中在Ndc80的MT结合区附近。Stu2在DAM1复合体着丝粒一侧的着丝粒中心约20 nm的狭义区域内定位于高丰度。我们的数据显示，发芽酵母着丝粒的微管附着部位组织得很好。Ndc80、DAM1和Stu2均沿动粒-MT轴在其平均位置附近狭窄分布。这些组分的相对组织、它们狭窄的分布以及它们已知的MT结合特性共同解释了它们的联合作用如何产生与MT聚合和解聚耦合的持久的、双向的动粒运动。
The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore. We used Förster Resonance Energy Transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed > 60 FRET and high resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ~ 20 nm on the centromeric side of the Dam1 complex. Our data show that the microtubule attachment site of the budding yeast kinetochore is well-organized. Ndc80, Dam1 and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.
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DOI：
10.1083/jcb.201209154
发表时间：
2013-02-18
期刊：
The Journal of cell biology
影响因子：
--
作者：
McIntosh JR;O'Toole E;Zhudenkov K;Morphew M;Schwartz C;Ataullakhanov FI;Grishchuk EL
通讯作者：
Grishchuk EL
DOI：
10.1073/pnas.0405925102
发表时间：
2005-04-12
影响因子：
11.1
作者：
Bouck, DC;Bloom, KS
通讯作者：
Bloom, KS
影响因子：
64.5
作者：
Ciferri, Claudio;Pasqualato, Sebastiano;Musacchio, Andrea
通讯作者：
Musacchio, Andrea
影响因子：
7.8
作者：
Lampert, Fabienne;Mieck, Christine;Westermann, Stefan
通讯作者：
Westermann, Stefan
DOI：
10.1016/j.cub.2009.02.056
发表时间：
2009-04-28
期刊：
Current biology : CB
影响因子：
--
作者：
Joglekar AP;Bloom K;Salmon ED
通讯作者：
Salmon ED