Tetrahymena Basal Body Duplication

中心体是由两个中心粒组成的微管组织中心（MTOC）， 中心粒周围物质精确的中心体复制需要每个细胞周期精确一次， 双极有丝分裂纺锤体的形成，负责染色体正确分离的装置 在有丝分裂期间。中心体缺陷导致遗传不稳定性，在某些癌症中很明显。中心粒 复制是中心体复制的关键事件，中心粒组装事件是 在机制上类似于另一种MTOC的那些，即使纤毛或鞭毛成核的基体。 中心粒和基体在结构上相似，共享许多组件，并且两者的组装 细胞器需要许多相同的基因。我们将研究纤毛类的基体组装 原生动物四膜虫。在中心粒和基体上发现的保守蛋白质中， centrin，一种小的钙结合EF手蛋白。我们研究了四膜虫的四个中心蛋白基因。的 这些，Cen 1定位于基体，并且Cen 1无效等位基因揭示基体组装中的缺陷， 上维护我们已经通过多维蛋白质鉴定了基底体的其他成分 识别技术（MudPIT）。该分析已经描述了超过50个候选基体 这些是我们要研究的内容。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成分将通过免疫电镜定位，以确定那些 定位于在基体组装中重要的结构。这些组件将受到遗传 分析，如Cen 1所做的，以确定它们是否确实在基体组装中起作用，或者 另一个功能在基体上。最后，体外组装测定的发展将有助于 详细了解中心粒复制。我们相信四膜虫非常适合这样一个 approach.我们已经建立了一个体外中心蛋白结合试验，导致识别α 和β-微管蛋白作为潜在的中心蛋白结合伙伴在基体，我们建议研究这一点， 互动我们还将描述一个体外系统，该系统形成了一个早期和重要的中间体， 基体组件。总的来说，我们的方法使用四膜虫将确定新的基体和中心粒 组件和定义的机制，他们的作用，基体和中心粒组装。

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.