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.

莱茵衣藻已成为真核细胞鞭毛器功能和发育分析的实验模型系统。基底器将鞭毛固定在细胞内，相当于哺乳动物中心体的功能。一些基础装置蛋白首先在绿色鞭毛虫中被发现，例如中心蛋白和 SF-组装蛋白。现在已知它们存在于许多其他真核生物中，在鞭毛/纤毛细胞中发挥重要功能。尽管轴丝的蛋白质组成和功能相对较好地了解，但仍然缺乏对基础装置的类似研究。基底器不仅参与鞭毛的形成，而且在缩瞳、胞质分裂和维持细胞极性方面发挥重要作用。参与这些基本细胞功能的基础装置蛋白很可能在整个进化过程中得到保守，并且对莱茵衣藻的基础装置亚蛋白质组的分析可能有助于识别哺乳动物和人类细胞中新的疾病相关基因。该项目旨在鉴定莱茵衣藻基础装置亚蛋白质组中的大部分或全部蛋白质。将分离和纯化基础装置，并通过质谱法进行高分辨率 2D-PAGE 鉴定蛋白质。对选定的新型基础装置蛋白的功能分析将涉及通过 RNAi 进行基因沉默，以及使用已建立的电穿孔方案通过蛋白质靶向进行功能拯救。最后，将使用 DNA 微阵列分析一系列基础装置蛋白的整体基因表达，重点是细胞周期、性分化和昼夜节律控制下基础装置亚蛋白质组的变化。