TETRAHYMENA BASAL BODY DUPLICATION

四膜虫基础体复制

• 批准号: 8362544
• 负责人: MARK WINEY
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362544
• 关键词: Biochemical; Cell physiology; Cells; Cellular Structures; Centrioles; Centrosome; Chemicals; Cytoplasm; Electrons; Freeze Substitution; Freezing; Funding; Future; Grant; Ice; In Vitro; Mechanics; Microtubule Triplet; Modeling; Morphology; National Center for Research Resources; Principal Investigator; Process; Reporting; Research; Research Infrastructure; Resources; Sampling; Site; Source; Structure; Tetrahymena; Tomogram; United States National Institutes of Health; cell fixing; cell type; cost; in vivo; kinetosome; mutant; pressure; sample fixation; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ciliate Tetrahymena is covered with more than 1000 basal bodies. Each basal body is composed of 9 triplet microtubules in a circular array, very similar in its organization to the centrioles in centrosomes. Also similar to centrioles, basal bodies are assembled from a specific site at the cellular end of an existing basal body. We have initiated an analysis of Tetrahymena basal body duplication as a model for centriole duplication. As part of this project, we have started electron tomographic analysis of basal bodies and their assembly intermediates in Tetrahymena. We have prepared Tetrahymena cells by chemical fixation and by high-pressure freezing and freeze-substitution. The basal bodies have excellent morphology with both these fixations, though some of the frozen cells show ice damage. A few preliminary tomograms of basal bodies in mutant and wild-type cells have been constructed to compare the two sample types. We have also done tomograms of basal bodies in pellicles. Pellicles are cell ghosts resulting from mechanical disruption of the cells and extraction of the cytoplasm. It has been previously reported that basal bodies remain intact in pellicles, and we have repeated that result. We are comparing the tomograms of basal bodies in intact cells and in pellicles, seeking any structural differences. We are using basal bodies in pellicles for localization and biochemical studies and will need to correlate those results with our structural studies of fixed cells. Our future plans for tomography of basal bodies in Tetrahymena include the description of basal body duplication in wild-type and in mutant cells. This analysis will help identify which assembly processes or intermediates are missing in the mutants. Furthermore, we will complete our tomographic examination of the basal bodies in pellicles. If we can assemble basal bodies or their intermediates in vitro, those structures will be examined by tomography to compare them with their in vivo counterparts.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 纤毛四膜虫由1000多个基体覆盖。 每个基体由9个三联微管组成，呈圆形排列，其组织结构与中心体中的中心粒非常相似。 与中心粒相似，基体也是从现有基体的细胞末端的特定位点组装而成。 我们已经开始分析四膜虫基体复制作为中心粒复制的模型。 作为这个项目的一部分，我们已经开始在四膜虫的基体及其组装中间体的电子断层扫描分析。 我们已经通过化学固定和高压冷冻和冷冻置换制备了四膜虫细胞。 在这两种固定条件下，基体具有良好的形态，尽管一些冷冻细胞显示出冰损伤。 已经构建了突变体和野生型细胞中的基底体的一些初步断层图像以比较两种样品类型。 我们还做了表膜基底体的断层扫描。 细胞膜是由细胞的机械破碎和细胞质的提取产生的细胞鬼。 以前曾报道过，基底体在表膜中保持完整，我们重复了这一结果。 我们正在比较完整细胞和表膜中基体的断层图像，寻找任何结构上的差异。 我们正在利用薄膜中的基体进行定位和生物化学研究，并将需要将这些结果与我们对固定细胞的结构研究相关联。 我们未来的计划，在四膜虫的基体断层扫描，包括在野生型和突变细胞的基体复制的描述。 这种分析将有助于确定突变体中缺少哪些组装过程或中间体。 此外，我们将完成我们的断层扫描检查的基体表膜。 如果我们能在体外组装基体或它们的中间体，这些结构将通过断层扫描进行检查，以将它们与体内对应物进行比较。