HVEM TOMOGRAPHY OF EPITOPE-TAGGED SPINDLE POLE BODY COMPONENTS

表位标记的主轴极体组件的 HVEM 断层扫描

• 批准号: 7354991
• 负责人: EILEEN T O TOOLE
• 金额: $ 0.47万
• 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/7354991
• 关键词: centrosome; face; fungal proteins; microtubules; mutant; organelles; protein engineering; proteins; sectioning; spindle pole body; success; tomography; 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. The spindle pole body (SPB) is the yeast centrosome and is the sole nucleation site for microtubules in this organism. Thus, the SPB is a critical organelle, serving as the pole of both mitotic and meiotic spindles. Electron tomography is a useful method to image the 3D fine structure of the yeast SPB (O'Toole, et al., 1999, Mol. Biol. Cell 10:2017-2031), so we are using this technique to obtain high-resolution morphological data. We are using image averaging techniques, previously developed in the Boulder 3D Laboratory, and new techniques developed with the help of Rick Gaudette, to visualize regular features of the SPB. For our first attempt, we have used a diploid strain expressing GFP-Spc42p construct. Spc42p forms a hexagonal crystal in a layer of the SPB immediately adjacent to the central plaque. This strain and a wild type control were high pressure frozen, freeze-substituted and embedded in Lowicryl HM20. To avoid artifacts due to section thinning, we developed a strategy to find face-on views of spindle pole bodies. Dual-axis tomograms have now been collected in these specimens and the regular array formed from the Spc42p can be identified, although the images are noisy. We are now pursing ways to average images of the crystalline layer of the SPB, thus mapping the position of the SPB component that contain the tag. With this approach, we hope to document additional structural features in the SPB, detect subtle changes in those features in mutants, and gain information on the position of SPB components. While this is technically difficult, the work is ongoing, because future success would greatly benefit our ability to localize protein components of ordered structures in systems like yeast, where protein engineering is feasible.

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。纺锤体极体（SPB）是酵母中心体，是该生物体中微管的唯一成核位点。因此，SPB 是一个重要的细胞器，充当有丝分裂和减数分裂纺锤体的极点。电子断层扫描是对酵母 SPB 3D 精细结构进行成像的有用方法 (O'Toole, et al., 1999, Mol. Biol. Cell 10:2017-2031)，因此我们使用该技术来获取高分辨率形态学数据。我们正在使用先前在 Boulder 3D 实验室开发的图像平均技术以及在 Rick Gaudette 的帮助下开发的新技术来可视化 SPB 的规则特征。对于我们的第一次尝试，我们使用了表达 GFP-Spc42p 构建体的二倍体菌株。 Spc42p 在紧邻中央斑块的 SPB 层中形成六方晶体。将该菌株和野生型对照进行高压冷冻、冷冻替代并包埋在 Lowicryl HM20 中。为了避免由于截面变薄而产生的伪影，我们制定了一种策略来查找主轴极体的正面视图。现在已经在这些样本中收集了双轴断层图，并且可以识别由 Spc42p 形成的规则阵列，尽管图像有噪声。我们现在正在寻找对 SPB 结晶层图像进行平均的方法，从而绘制包含标签的 SPB 组件的位置。通过这种方法，我们希望记录 SPB 中的其他结构特征，检测突变体中这些特征的细微变化，并获得有关 SPB 组件位置的信息。虽然这在技术上很困难，但这项工作仍在继续，因为未来的成功将极大地有利于我们在酵母等系统中定位有序结构的蛋白质成分的能力，在这些系统中蛋白质工程是可行的。