MOLECULAR ANALYSIS OF CEP135 IN THE CENTROSOME

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许多 严重的人类疾病，包括出生缺陷和癌症。钥匙 结构到微管功能的机制是MTOC (微管组织中心)，或中心体，由一个 一对中心粒和周围无定形的中心粒周围 材料。中心体控制着时间和空间分布 以及最近的证据表明，癌细胞 包括结构和功能异常的中心体。目的 我们的研究是为了了解中心体如何调节微管 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(Cep135)，存在于 种类繁多的生物。它是一种结构蛋白，参与了 有丝分裂过程中的微管组织和纺锤体组装。 我们建议通过实验来扩展我们对Cep135的研究，以便 评估工作假设，即Cep135是 中心体对于维持心脏的整体完整性很重要 中央三极周围物质。功能分析将使用两种方法进行 稳定表达不同形式Cep135和Cep135的CHO细胞株 非洲爪哇无细胞萃取物。Cep135在血管紧张素转换酶激活过程中的作用 两极纺锤体的形成将在p53缺失的细胞中进行评估 含有中心体的多个副本。Cep135结果的过度表达 在形成特殊的丝状结构的过程中 中心体；由变化引起的形态缺陷的性质 Cep135的水平将使用高分辨率来确定 免疫荧光显微镜和电子断层扫描。最后，我们会 研究Cep135与其他分子的相互作用模式 中心体和一个大的含有Cep135的组分在 青蛙提取物将进一步表征。

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.