Tetrahymena Basal Body Duplication

中心体是由两个中心粒组成的微管组织中心，中心粒被 中央三极周围物质。需要在每个细胞周期内精确复制一次中心体 两极有丝分裂纺锤体的形成，负责染色体的适当分离 在有丝分裂期间。中心体缺陷会导致遗传不稳定，在某些癌症中很明显。中心粒 复制是中心体复制中的关键事件，中心粒组装事件是 在力学上类似于另一种MTOC，即形成纤毛或鞭毛的基底体。 中心粒和基本体在结构上相似，有许多共同的成分，两者的组装 细胞器需要许多相同的基因。我们将研究纤毛虫的基底体组装 原生动物嗜热四膜虫。在中心粒和基体处发现的保守蛋白质中有 Centin，一种小的钙结合的EF手蛋白。我们研究了四膜虫的四个中心素基因。的 这些，Cen1定位于基底体，CEN1零等位基因显示基底体组装和 维修。我们已经通过多维蛋白质鉴定了基底的其他成分 身份识别技术(MudPIT)。这一分析已经描述了50多个候选基底体 我们建议研究的组件。最初，我们将定位标记的蛋白质版本以确认 它们是身体的基本组成部分。新的组件将通过免疫-EM进行本地化，以识别 定位于在基础身体组装中重要的结构。这些成分将受到遗传基因的影响 分析，如对Cen1所做的，以确定它们是否确实在基础机体组装中发挥作用 基础身体的另一种功能。最后，体外组装试验的发展将有助于 详细了解中心粒复制。我们认为四膜虫非常适合于这样的 接近。我们已经建立了一种体外中心素结合试验，该试验已经导致了阿尔法的鉴定 和β-微管蛋白作为基底体潜在的中心蛋白结合伙伴，我们建议对此进行研究 互动。我们还将描述形成早期和重要中间产物的体外系统的特征。 基本车身组件。总体而言，我们使用四膜虫的方法将识别新的基底和中心粒。 并确定它们在基本体和中心粒组装中的作用机制。

Centrosomes are microtubule-organizing centers (MTOCs) consisting of two centrioles surrounded by pericentriolar material. Accurate centrosome duplication precisely once per cell cycle is required for formation of a bipolar mitotic spindle, the apparatus responsible for proper segregation of chromosomes during mitosis. Centrosome defects cause genetic instability and are evident in certain cancers. Centriole duplication is a critical event in centrosome duplication, and the events of centriole assembly are mechanistically similar to those of another MTOC, the basal body, which nucleates cilia or flagella. Centrioles and basal bodies are similar in structure, share many components, and the assembly of the two organelles require many of the same genes. We will investigate basal body assembly in the ciliated protozoan Tetrahymena thermophila. Among the conserved proteins found at centrioles and basal bodies is centrin, a small calcium-binding EF hand protein. We have studied four centrin genes in Tetrahymena. Of these, Cen1 localizes to basal bodies, and a CEN1 null allele reveals defects in basal body assembly and maintenance. We have identified additional components of basal bodies by Multidimensional Protein Identification Technololgy (MudPIT). This analysis has described well over 50 candidate basal body components which we propose to study. Initially, we will localize tagged versions of the proteins to confirm that they are basal body components. New components will be localized by immuno-EM to identify those that localize to structures important in basal body assembly. These components will be subjected to genetic analysis, as done with Cen1, to determine whether they indeed have a role in basal body assembly or have another function at the basal body. Finally, the development of an in vitro assembly assay will contribute to a detailed understanding of centriole duplication. We believe that Tetrahymena is ideally suited for such an approach. We have established an in vitro centrin binding assay that has led to the identification of alpha and beta-tubulin as potential centrin binding partners at basal bodies, and we propose to study this interaction. We also will characterize an in vitro system that forms an early and important intermediate in basal body assembly. Overall, our approaches using Tetrahymena will identify new basal body and centriole components and define mechanisms for their roles in basal body and centriole assembly.