HVEM TOMOGRAPHY OF EPITOPE-TAGGED SPINDLE POLE BODY COMPONENTS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。纺锤体是酵母的中心体，也是该生物体中微管的唯一成核部位。因此，SPB是一个重要的细胞器，是有丝分裂纺锤体和减数分裂纺锤体的极。电子断层扫描是对酵母SPB的3D精细结构进行成像的有用方法(O‘Toole等人，1999，Mol.比奥尔。细胞10：2017-2031)，所以我们正在使用这项技术来获得高分辨率的形态数据。我们正在使用之前在博尔德3D实验室开发的图像平均技术，以及在里克·高德特的帮助下开发的新技术，来可视化SPB的常规特征。在我们的第一次尝试中，我们使用了一个表达GFP-Spc42p的二倍体菌株。Spc42P在紧邻中央斑块的SPB层中形成六方晶体。将该菌株和一个野生型对照菌株进行高压冷冻、冷冻置换和包埋在Lowicryl HM20中。为了避免由于截面变薄而产生的伪影，我们开发了一种策略来找到主轴极体的面对面视图。现在已经在这些标本中收集了双轴断层图像，可以识别由Spc42P形成的规则阵列，尽管图像有噪声。我们现在正在寻找方法来平均SPB的结晶层的图像，从而映射包含标签的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，并获得关于SPB组件位置的信息。虽然这在技术上是困难的，但这项工作仍在进行中，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。

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.