TETRAHYMENA BASAL BODY DUPLICATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 纤毛虫四膜虫被1000多个基体所覆盖。每个基底体由9个三联体微管组成圆形阵列，其组织结构与中心体中的中心粒非常相似。同样与中心粒相似，基底体也是从现有基底体的细胞端的特定位置组装而成的。我们已经开始分析四膜虫基本体复制作为中心粒复制的模型。作为该项目的一部分，我们已经开始对四膜虫的基本体及其组装中间体进行电子断层扫描分析。我们用化学固定和高压冷冻、冷冻替代的方法制备了四膜虫细胞。在这两种固定方法下，基底小体的形态都很好，尽管一些冷冻细胞显示出冰冻的损伤。对突变型和野生型细胞中的基底体进行了一些初步的断层扫描，以比较这两种样本类型。我们还做了膜基底体的断层摄影。细胞膜是由于细胞的机械破坏和细胞质的提取而产生的细胞幽灵。此前有报道称，基底小体在细胞膜中保持完好，我们重复了这一结果。我们正在比较完整细胞和细胞膜中的基底体的断层图像，以寻找任何结构上的差异。我们正在利用细胞膜中的基底体进行定位和生化研究，并需要将这些结果与我们对固定细胞的结构研究相关联。 我们未来计划对四膜虫的基底体进行断层扫描，包括描述野生型和突变型细胞的基底体复制。这项分析将有助于确定突变体中缺少哪些组装过程或中间产物。此外，我们还将完成对透明膜内基底体的断层检查。如果我们能够在体外组装基底或它们的中间体，这些结构将通过断层扫描进行检查，以便与体内的对应结构进行比较。

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 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.