STRUCTURAL INTEGRITY OF CENTROSOME IN MAMMALIAN CELLS

（由烟草研究公司理事会支持。#31572;的R。 Kuriyama）中心体，由一对中心粒组成 和周围的无定形云的中心粒周围的物质， 在微管组织中的重要作用。 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。 我们试图将这种蛋白质定位在中心体中， 免疫电子显微镜下观察到的中心粒周围物质。 然后我们计划 评估Cep 135与其他 中心体中的亚结构，包括γ-微管蛋白环，基底 英尺和卫星，采用高压和中压 电子显微镜断层扫描，已被证明是有效的， 定义了几个域，包括在定义不清的中心粒周围 材料 初步观察已证明， Cep 135亚结构域的过表达导致显著的修饰 的中心体结构和稳定性。 到 评估Cep 135亚结构域在结构完整性中的作用 中心体，我们将创建一系列缺失结构， 在CHO细胞中表达Cep 135的不同区域。 详细性质 中心体在形态和功能上的变化 将在体内和体外进行检查。 栗山博士的实验室 制备EM块以及细胞的薄片显微照片 感兴趣 牢房的位置也标在街区上。 连续切下0.25和0.5 μ m厚的切片，并在 HVEM。 较薄的部分被证明更适合定位 中心粒 对两组细胞的中心粒进行拍照， 底片被送到栗山博士那里进行中心粒的选择 用于断层扫描。

(Supported by Council for Tobacco Research Inc. #3157 R2 to R. Kuriyama) The centrosome, which is composed of a pair of centrioles and a surrounding amorphous cloud of pericentriolar material, plays an essential role in 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 are attempting to localize this protein in the pericentriolar material by immunoelectron microscopy. We then plan to assess the temporal and spatial relation of Cep135 with other substructures in the centrosome, including gamma-tubulin rings, basal feet and satellites, 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 pr ovided e vidence that over-expression of Cep135 subdomains results in striking modifications of the centrosomal structure and stability in transfected cells. To evaluate the role of Cep135 subdomains 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. Dr. Kuriyama's lab prepared EM blocks together with thin-section micrographs of the cells of interest. The cell locations were marked on the blocks, as well. Serial 0.25 and 0.5(m thick sections were cut and examined on the HVEM. The thinner sections proved to be better for locating the centrioles. Centrioles were photographed from two groups of cells and the negatives were sent to Dr. Kuriyama for selection of centrioles for tomography.