STRUCTURAL INTEGRITY OF CENTROSOME IN MAMMALIAN CELLS

中心体由一对中心粒和一个 围绕着中心粒周围物质的无定形云， 微管组织的重要作用。 我们最近 发现了一种新的中心体蛋白，称为Cep 135， 在有丝分裂过程中对纺锤体的形成和功能很重要。 到 理解Cep 135在组织结构中的作用。 中心体，我们将首先分析的详细定位的 免疫电镜观察到的中粒周物质中的蛋白。 Cep 135与其他亚结构的时空关系 中心体，包括γ-微管蛋白环，基底足和 卫星，下一步将评估采用高， 中压电子显微镜断层扫描已经证明 为了有效地界定几个领域， 中心粒周围物质 初步观察显示， 有证据表明Cep 135亚结构域的过度表达导致艾德 中心体结构和稳定性的显着修改， 转染细胞 为了评估Cep 135亚结构域在 中心体的结构完整性，我们将创建一系列 缺失构建体在CHO细胞中表达Cep 135的各个区域。 形态学和功能学的详细性质 中心体的改变将在体内和体内进行检查。 体外 八个街区被送去进行初步研究。 切片 切割并染色（0.25和0.5微米），并在HVEM上观察。 的 细胞数量众多，定位中心粒很耗时。 中心粒在0.25微米切片中的图像看起来非常清晰。 “泥泞”。 精细结构不清楚。 还观察了网格 在蔡司910和记录的图像仍然缺乏清晰的 详细 我们讨论了样品制备导致 中心粒的“浑浊”外观，以及我们的完整程序 用于处理和平包埋生长在盖玻片上的细胞， 送给栗山医生 我们没能看到任何“螺旋状” 与中心粒相关的结构在这个初步的 考试

The centrosome, which is composed of a pair of centrioles and a surrounding amorphous cloud of pericentriolar material, plays an essential role for microtubule organization. We have recently identified a novel centrosomal protein, termed Cep135, which appears to be important for spindle formation and function during mitosis. To understand the role of Cep135 in the structural organization of the centrosome, we will first analyze the detailed localization of the protein in the pericentriolar material by immuno-electron microscopy. Temporal and spatial relation of Cep135 with other substructures in the centrosome, including gamma-tubulin rings, basal feet and satellites, will next be assessed by employing both high and intermediate voltage electron microscope tomography, which has proven to be effective in defining several domains included in ill-defined pericentriolar material. Preliminary observation has provided evidence that over expression of Cep135 sub-domains result ed in striking modifications of the centrosomal structure and stability in transfected cells. To evaluate the role of Cep135 sub-domains in the structural integrity of the centrosome, we will create a series of deletion constructs to express various regions of Cep135 in CHO cells. The detailed nature of morphological as well as functional alternations of the centrosome will be examined both in vivo and in vitro. Eight block were sent for preliminary studies. Sections were cut and stained (0.25 and 0.5 micron) and viewed on the HVEM. The cells are numerous and the location of centrioles is time consuming. The images of the centrioles in 0.25 micron sections appear very "muddy". The fine structure is not clear. The grids were also looked at on the Zeiss 910 and the recorded images are still lacking in clear detail. We discussed the possibility of sample preparation causing the "muddy" appearance of the centrioles, and our complete procedure for processing and flat embeddment of cells grown on coverslips was sent to Dr. Kuriyama. We were not able to see any "whorl-like" structures associated with the centrioles in this preliminary examination.