The basal apparatus sub-proteome of Chlamydomonas reinhardtii - composition, gene expression and functional analysis

莱茵衣藻的基础装置亚蛋白质组 - 组成、基因表达和功能分析

• 批准号: 5402429
• 负责人: Professor Dr. Michael Melkonian
• 金额: --
• 依托单位: Abteilung Pflanze-Mikroben Interaktionen
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5402429?language=en
• 关键词: basal; apparatus; sub; proteome; Chlamydomonas

Chlamydomonas reinhardtii has become the experimental model system for the functional and developmental analysis of the flagellar apparatus in eukaryotic cells. The basal apparatus anchors the flagella inside the cell and represents the functional equivalent of the mammalian centrosome. Several basal apparatus proteins have been first identified in green flagellates such as centrin and SF-assemblin. They are now known to occur in many other eukaryotic organisms performing essential functions in flagellate/ciliate cells. Whereas the protein composition and function of the axoneme are relatively well understood, comparable studies on the basal apparatus are still lacking. The basal apparatus is not only involved in the formation of the flagella, but also plays important roles in miosis, cytokinesis, and in maintaining cell polarity. It is likely that basal apparatus proteins involved in these essential cellular functions were conserved throughout evolution and that analysis of the basal apparatus sub-proteome of C. reinhardtii may help to identify novel disease-related genes in mammalian and human cells. The project aims to identify most or all of the proteins in the basal apparatus sub-proteome of C. reinhardtii. Basal apparatuses will be isolated, and purified and proteins will be identified following high-resolution 2D-PAGE by mass spectrometry. Functional analyses of selected novel basal apparatus proteins will involve gene silencing by RNAi, and functional rescue by protein targeting using an established electroporation protocol. Finally, global gene expression of a selection of basal apparatus proteins using DNA-microarrays will be analyzed with emphasis on changes in the basal apparatus sub-proteome during cell cycle, sexual differentiation and under circadian control.

衣原体Reinhardtii已成为真核细胞中鞭毛装置功能和发育分析的实验模型系统。基部设备将鞭毛锚定在细胞内部，并代表哺乳动物中心体的功能性。首先在绿色鞭毛蛋白（例如中心蛋白和SF囊膜）中鉴定出几种基底蛋白。现在已知它们发生在许多其他真核生物中，在鞭毛/纤毛细胞中执行基本功能。尽管轴突的蛋白质组成和功能相对众所周知，但仍缺乏对基础设备的可比研究。基础设备不仅参与鞭毛的形成，而且在MIOSIS，细胞因子和维持细胞极性方面起着重要作用。在整个进化过程中，参与这些基本细胞功能的基础设备蛋白很可能是保守的，并且对Reinhardtii的基础仪器亚蛋白质的分析可能有助于鉴定哺乳动物和人类细胞中与疾病相关的新型基因。该项目旨在鉴定Reinhardtii的基底仪器亚蛋白质中的大多数或所有蛋白质。将分离基础设备，并在高分辨率的2d-page通过质谱时识别纯化和蛋白质。选定的新型基础设备蛋白的功能分析将涉及RNAi的基因沉默，以及使用已建立的电穿孔方案靶向蛋白质的功能拯救。最后，将分析使用DNA微阵列的基础设备蛋白选择的全球基因表达，重点是在细胞周期，性别分化和昼夜节律控制下基础设备亚蛋白蛋白的变化。