KINETOCHORES IN THE FISSION YEAST, SCHIZOSACCHAROMYCES POMBE

裂殖酵母中的着丝粒，裂殖酵母

• 批准号: 7355019
• 负责人: XIANGWEI HE
• 金额: $ 1.41万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355019
• 关键词: centromere; yeasts

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. By tandem-affinity-purification (TAP), the He lab has purified three major kinetochore protein complexes from fission yeast (Liu, 2005). Among them, the NMS and Sim4 complexes are constitutive elements of a core kinetochore structure. By metal shadowing EM, purified NMS and Sim4 complexes are both seen as fibrils (He X., unpublished). Fibrils are commonly observed by EM at several types of kinetochores (e.g. McEwen et al., 1998 and McIntosh J.R., unpublished results). While the protein sequence conservation of kinetochore components is not strong, it is possible that the common structural features (fibrils in this case), rather than sequence similarity in individual proteins, provides the basis for a ubiquitous mechanism that is shared by all types of kinetochores. One tool now available for the studying the functional significance of the NMS complex is a temperature sensitive allele of one of its subunits, nuf2-1. Kinetochores in nuf2-1 cells grown at their restrictive temperature fail to attach to the spindle, (Nabetani et al., 2001). However, under the same condition, the NMS complex remains intact and is properly localized on the kinetochores (He, X. unpublished). We will determine whether the NMS fibrils are still found at kinetochores and whether there is a change in their structure or position relative to the kMTs. The third kinetochore complex, Dam1, has been purified only from mitotic extracts (Liu, 2005). In vivo, the Dam1 complex associates with kinetochores and spindle microtubules specifically in mitosis. A recombinant form of budding yeast Dam1 complex was shown to assemble a ring around MTs in vitro (Miranda et al., 2005; Westermann et al., 2005). However, no such ring structure around kMTs has yet been seen in vivo. We will use a combination of the immuno-gold labeling and EM tomography to determine the localization of the Dam1 complex relative to the kMTs and provide the most powerful evidence about the presence or the absence of a Dam1 ring. Using Immuno-EM and EM tomography of cells with GFP tags on 3 of the Dam1 subunits, the Dam1 complex can be localized in thin (70nm) sections, and any possible fine structural features, such as rings, that are associated with the Dam1 complex will be sought within the section.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。通过串联亲和纯化（TAP），He 实验室从裂殖酵母中纯化了三种主要的动粒蛋白复合物（Liu，2005）。其中，NMS和Sim4复合体是核心动粒结构的组成元件。通过金属阴影电镜，纯化的 NMS 和 Sim4 复合物都被视为原纤维（He X.，未发表）。电镜通常在几种类型的动粒处观察到原纤维（例如 McEwen 等人，1998 年和 McIntosh J.R.，未发表的结果）。虽然着丝粒成分的蛋白质序列保守性并不强，但共同的结构特征（在本例中为原纤维），而不是单个蛋白质的序列相似性，可能为所有类型的着丝粒共享的普遍机制提供了基础。现在可用于研究 NMS 复合体功能意义的一种工具是其亚基之一 nuf2-1 的温度敏感等位基因。在限制温度下生长的 nuf2-1 细胞中的动粒无法附着在纺锤体上（Nabetani 等，2001）。然而，在相同条件下，NMS 复合体保持完整并正确定位在着丝粒上（He，X. 未发表）。我们将确定着丝粒处是否仍存在 NMS 原纤维，以及它们相对于 kMT 的结构或位置是否发生变化。第三个动粒复合体 Dam1 仅从有丝分裂提取物中纯化（Liu，2005）。在体内，Dam1 复合物与着丝粒和纺锤体微管相关，特别是在有丝分裂中。出芽酵母 Dam1 复合体的重组形式被证明可以在体外围绕 MT 组装成一个环（Miranda 等人，2005 年；Westermann 等人，2005 年）。然而，在体内尚未发现 kMT 周围的这种环状结构。我们将结合使用免疫金标记和 EM 断层扫描来确定 Dam1 复合物相对于 kMT 的定位，并提供有关 Dam1 环是否存在的最有力证据。使用免疫-EM 和 EM 断层扫描对 3 个 Dam1 亚基上带有 GFP 标签的细胞进行成像，可以将 Dam1 复合物定位在薄 (70 nm) 切片中，并且将在该切片内寻找与 Dam1 复合物相关的任何可能的精细结构特征，例如环。