Protein topology at the Dictyostelium centrosome

盘基网柄菌中心体的蛋白质拓扑

• 批准号: 394020894
• 负责人: Dr. Irene Meyer
• 金额: --
• 依托单位: Institut für Biochemie und Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394020894?language=en
• 关键词: Protein; topology; Dictyostelium; centrosome

Centrosomes are the main organizers of microtubules and therefore they play a crucial role in the maintenance of cell architecture and cell cycle progression. They root back to the last eukaryotic common ancestor. Hence they are found in most eukaryotic supergroups, although with rather divergent appearances. Organisms harboring cilia or flagellae in at least one cell type possess centrosomes with centrioles, which are also capable of functioning as basal bodies in cilia formation. In contrast, organisms lacking cilia for locomotion such as amoebozoans or many fungi, usually contain centrosomes without centrioles. As a representative of the latter group, we investigate the centrosome (also called nucleus-associated body) in Dictyostelium amoebae, a very useful species to study those centrosomal functions that are independent of centrioles. Rather than having centrioles, the spheroid Dictyostelium centrosome consists of a three-layered core structure, embedded in a matrix containing microtubule-nucleation complexes, the corona. We have characterized most if not all structural centrosomal components on a molecular level. However, while most individual components have been successfully allocated either to the core or the corona structure, the elucidation of subcentrosomal protein topology and mutual protein interactions is still in its infancy. In our work program we will address the following questions: - Which protein(s) mediate the interaction of the outer layers of the core structure with the corona?- Which protein(s) mediate the interaction of the outer layers with the central layer?- Which proteins interact with the -tubulin-containing nodules within the corona, and/or act as scaffolding proteins?To answer these questions, we will combine proximity-dependent biotin identification (BioID) to map protein-protein interactions on the biochemical level, with superresolution light microscopy (STED and dSTORM) and electron microscopy to draw a detailed topological map of protein localizations at the centrosome. In order to circumvent the need for highly specific antibodies for each protein, and to improve the resolution of our microscopic methods, we will create knock-in strains in which the endogenous centrosomal proteins of interest are completely replaced with tagged variants expressed under control of the endogenous promoter. These tags will be visualized by fluorescent tag-specific nanobodies, or, in case of EM, with Ni-NTA nanogold or gold-labeled nanobodies.

中心体是微管的主要组织者，因此它们在维持细胞结构和细胞周期进程中起着至关重要的作用。它们可以追溯到最后一个真核生物的共同祖先。因此，它们存在于大多数真核生物的超群中，尽管它们的外观相当不同。在至少一种细胞类型中具有纤毛或鞭毛的生物体具有中心粒的中心体，中心粒也能够在纤毛形成中起基体的作用。相比之下，缺乏纤毛的生物体，如变形虫或许多真菌，通常含有中心体而没有中心粒。作为后一组的代表，我们调查的中心体（也称为核相关体）在Dictyosteelium amoeelium，一个非常有用的物种，以研究这些中心体的功能，是独立的中心粒。而不是有中心粒，球状网柄藻中心体由一个三层的核心结构，嵌入在一个矩阵包含微管成核复合物，冠。我们已经在分子水平上表征了大多数（如果不是全部的话）结构中心体组分。然而，虽然大多数个别组件已成功地分配到核心或冠状结构，亚中心体蛋白拓扑结构和相互蛋白质相互作用的阐明仍处于起步阶段。在我们的工作计划中，我们将解决以下问题：-哪些蛋白质介导核心结构外层与冠层的相互作用？-哪种蛋白质介导外层与中心层的相互作用？哪些蛋白质与冠内含有微管蛋白的结节相互作用，和/或充当支架蛋白？为了回答这些问题，我们将结合联合收割机邻近依赖性生物素识别（BioID），在生物化学水平上绘制蛋白质-蛋白质相互作用，超分辨率光学显微镜（STED和dSTORM）和电子显微镜绘制详细的蛋白质定位在中心体的拓扑图。为了避免对每种蛋白质的高度特异性抗体的需要，并提高我们的显微镜方法的分辨率，我们将创建敲入菌株，其中感兴趣的内源性中心体蛋白质被内源性启动子控制下表达的标记变体完全取代。这些标签将通过荧光标签特异性纳米抗体显现，或者在EM的情况下，用Ni-NTA纳米金或金标记的纳米抗体显现。