MOLECULAR ANALYSIS OF CEP135 IN THE CENTROSOME

中心体中 CEP135 的分子分析

• 批准号: 6019276
• 负责人: RYOKO KURIYAMA
• 金额: $ 20.29万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019276
• 关键词: CHO cells; Xenopus oocyte; affinity chromatography; cell cycle; cell free system; centrosome; fluorescence microscopy; immunoelectron microscopy; laboratory mouse; laboratory rabbit; laboratory rat; microtubules; mitotic spindle apparatus; molecular biology; nuclear matrix; orientation; protein binding; protein biosynthesis; protein structure function; tomography; tumor suppressor proteins; video microscopy

Microtubules are involved in fundamental cellular processes, such as mitosis/meiosis, cell motility, intracellular transport and morphogenesis. Any perturbation in their functions causes a number of serious human diseases including birth defects and cancers. The key structure to the mechanism of the microtubule function is the MTOC (microtubule-organizing center), or centrosome which is composed of a pair of centrioles and the surrounding amorphous pericentriolar material. The centrosome controls the temporal and spatial distribution of microtubules, and recent evidence has indicated that cancer cells include centrosomes abnormal in structure and function. The purpose of our research is to understand how the centrosome regulates microtubule nucleation, orientation and anchorage at the molecular level. We have recently identified a novel centrosomal component (Cep135) present in a wide range of organisms. It is a structural protein involved in microtubule organization and spindle assembly during mitosis. Experiments are proposed to extend our studies on Cep135 in order to evaluate the working hypothesis that Cep135 is a matrix component of the centrosome important for maintenance of overall integrity of the pericentriolar material. Functional analysis will be done using both stable CHO cell lines expressing different forms of Cep135 and the Xenopus cell free extracts. The role of Cep135 in the process of bipolar spindle formation will be assessed in p53-deficient cells which contain multiple copies of centrosomes. Overexpression of Cep135 results in the formation of extraordinary filamentous structures in the centrosome; the nature of morphological defects caused by the change in the level of Cep135 will be determined using high resolution immunofluorescence microscopy and electron tomography. Finally, we will investigate the pattern of Cep135 interaction with other molecules in the centrosome, and a large Cep135-containing fraction identified in frog extracts will be further characterized.

微管参与基本的细胞过程，如 有丝分裂/减数分裂，细胞运动，细胞内运输和 形态发生 它们的功能中的任何扰动都会导致一些 严重的人类疾病，包括先天缺陷和癌症。 关键 微管功能的机制是MTOC （微管组织中心），或中心体，它是由一个 一对中心粒和周围无定形的中心粒周 材料 中心体控制着时空分布 最近的证据表明，癌细胞 包括结构和功能异常中心体。 的目的 我们的研究是为了了解中心体如何调节微管， 在分子水平上的成核、取向和锚定。 我们有 最近发现了一种新的中心体成分（Cep 135），存在于 各种各样的有机体。 它是一种结构蛋白， 微管组织和纺锤体组装。 实验提出了扩大我们的研究Cep 135，以便 评估Cep 135是Cep 135的基质成分的工作假设。 中心体对于维持细胞的整体完整性很重要， 中心粒周围物质 功能分析将使用 表达不同形式的Cep 135的稳定CHO细胞系， 爪蟾无细胞提取物。 Cep 135在这一过程中的作用 将在p53缺陷细胞中评估双极纺锤体的形成， 含有多个中心体拷贝。Cep 135过表达结果 形成了非凡的丝状结构 中心体的性质的变化所造成的形态缺陷， Cep 135的水平将使用高分辨率来确定 免疫荧光显微术和电子断层扫描。 最后我们将 研究Cep 135与其他分子相互作用的模式， 中心体，和一个大的Cep 135含有部分确定在 青蛙提取物将被进一步表征。