Recognizing chromosomes in trouble: Association of the spindle checkpoint protein Bub3p with altered kinetochores and a unique defective centromere
Recognizing chromosomes in trouble: Association of the spindle checkpoint protein Bub3p with altered kinetochores and a unique defective centromere
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DOI：
10.1128/mcb.23.18.6406-6418.2003
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发表时间：
2003-09-01
影响因子：
5.3
通讯作者：
Basrai, MA
中科院分区：
文献类型：
--
作者：
Kerscher, O;Crotti, LB;Basrai, MA
纺锤体检查点蛋白监测纺锤体与动粒的相互作用，即使只有一条染色体的微管附着被改变，也会停止后期。在这项研究中，我们表明，Bub 3 p的酿酒酵母，一个进化上保守的纺锤体检查点蛋白，表现出不同的相互作用与改变或有缺陷的动粒（S）。我们首次证明了绿色荧光蛋白标记的S.酿酒酵母Bub 3 p（Bub 3-GFP）不仅表现出弥散的核定位模式，而且在未受干扰的生长和G（2）/M停滞的细胞中形成明显的核灶。由于Bub 3-GFP焦点仅与动粒的一个子集重叠，因此我们测试了一个模型，其中动粒或纺锤体完整性的改变或缺陷导致Bub 3 p在这些结构上明显富集。为了支持我们的模型，着丝粒相关的Bub 3-GFP在纺锤体检查点激活后富集，这是由于诺考达唑诱导的纺锤体解体、检查点激酶Mps 1 p的过表达或缺陷性着丝粒（CEN）的存在。最重要的是，使用染色质免疫沉淀（ChIP）技术和基因工程缺陷CEN [CF/CEN 6（Delta 31）]的新方法，我们确定Bub 3-GFP可以与单个缺陷动粒相关。我们的研究代表了在野生型或有缺陷的动粒背景下，通过使用活细胞成像和S.啤酒。
Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.