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

莱茵衣藻已成为分析真核细胞鞭毛器功能和发育的实验模型系统。基器将鞭毛固定在细胞内，在功能上相当于哺乳动物的中心体。在绿色鞭毛中首次发现了几种基器蛋白，如Centin和SF-Assembly in。现已知，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。虽然轴丝的蛋白质组成和功能已被较好地了解，但对基础器官的可比性研究仍然缺乏。基器不仅参与鞭毛的形成，而且在减数分裂、胞质分裂和维持细胞极性方面起着重要作用。参与这些基本细胞功能的基器蛋白很可能在整个进化过程中都是保守的，对莱茵梭菌基器亚蛋白质组的分析可能有助于在哺乳动物和人类细胞中发现与疾病相关的新基因。该项目的目的是鉴定莱茵梭子蟹基本器官亚蛋白质组中的大部分或全部蛋白质。基础器官将被分离和纯化，并将在高分辨率2D-PAGE后通过质谱学鉴定蛋白质。对选定的新的基础器官蛋白的功能分析将涉及通过RNAi进行基因沉默，以及通过使用已建立的电穿孔方案通过蛋白质靶向进行功能挽救。最后，将使用DNA微阵列分析一系列基础器官蛋白质的整体基因表达，重点是基本器官亚蛋白质组在细胞周期、性分化和昼夜节律控制下的变化。

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.