KINETOCHORES IN THE FISSION YEAST, SCHIZOSACCHAROMYCES POMBE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。通过串联亲和纯化（TAP）， He实验室从裂变酵母中纯化了三种主要的着丝点蛋白复合物（Liu, 2005）。其中，NMS和Sim4复合体是核心着丝点结构的组成要素。通过金属遮蔽EM，纯化的NMS和Sim4配合物都被视为原纤维（He X，未发表）。原纤维通常在几种类型的着丝点上被电镜观察到（例如McEwen等人，1998年和McIntosh j.r.，未发表的结果）。虽然着丝点成分的蛋白质序列保守性不强，但可能是共同的结构特征（在这种情况下是原纤维），而不是单个蛋白质的序列相似性，为所有类型的着丝点共享的普遍机制提供了基础。目前研究NMS复合体功能意义的一个工具是其亚基之一nuf2-1的温度敏感等位基因。在限制温度下生长的nuf2-1细胞中的着丝点不能附着在纺锤体上（Nabetani et al, 2001）。然而，在相同的条件下，NMS复合体保持完整，并适当地定位在着丝点上（He， X.未发表）。我们将确定NMS原纤维是否仍然存在于着丝点，以及它们的结构或相对于kmt的位置是否发生了变化。第三个着丝点复合体，Dam1，仅从有丝分裂提取物中纯化（Liu, 2005）。在体内，Dam1复合体在有丝分裂中特异地与着丝点和纺锤体微管结合。一种重组形式的出芽酵母Dam1复合体被证明可以在体外围绕mt组装一个环（Miranda等人，2005；Westermann等人，2005）。然而，在体内尚未发现这种围绕kmt的环状结构。我们将结合免疫金标记和EM断层扫描来确定Dam1复合物相对于kmt的定位，并提供关于Dam1环存在或不存在的最有力证据。使用在3个Dam1亚基上带有GFP标签的细胞的免疫-EM和EM断层扫描，Dam1复合体可以定位在薄（70nm）的切片上，并且可以在切片中寻找与Dam1复合体相关的任何可能的精细结构特征，如环。

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 By metal shadowing EM, purified NMS and Sim4 complexes are both seen as fibrils (He X., unpublished). Fibrils are commonly observed by EM at several types of kinetochores (e.g. McEwen et al., 1998 and McIntosh J.R., unpublished results).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However, under the same condition, the NMS complex remains intact and is properly localized on the kinetochores (He, X. unpublished).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.