Protein topology at the Dictyostelium centrosome

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的真核生物共同祖先。因此，它们在大多数真核生物超群中都有发现，尽管它们具有相当不同的外观。在至少一种细胞类型中含有纤毛或鞭毛的生物体具有具有中心粒的中心体，中心粒也能够在纤毛形成过程中起基底体的作用。相比之下，缺乏纤毛的生物体，如变形虫或许多真菌，通常含有没有中心粒的中心体。作为后者的代表，我们研究了变形虫Dictyostelium amoebae的中心体（也称为核相关体），这是一个非常有用的物种，研究那些独立于中心粒的中心体功能。球形盘基骨中心体没有中心粒，而是由三层核心结构组成，嵌入含有微管成核复合物的基质中，即日冕。我们已经在分子水平上描述了大多数（如果不是全部）中心体结构成分。然而，虽然大多数单独的成分已经成功地分配到核心或冠结构，亚中心体蛋白质拓扑结构和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：-哪种蛋白质介导核心结构外层与日冕的相互作用？-哪些蛋白质介导了外层与中间层的相互作用？-哪些蛋白质与冠状内含有-微管蛋白的结节相互作用，和/或作为支架蛋白？为了回答这些问题，我们将结合邻近依赖生物素鉴定（BioID）在生化水平上绘制蛋白质-蛋白质相互作用，并使用超分辨率光学显微镜（STED和dSTORM）和电子显微镜绘制中心体蛋白质定位的详细拓扑图。为了避免对每种蛋白质的高度特异性抗体的需求，并提高显微镜方法的分辨率，我们将创建敲入菌株，其中内源性中心体蛋白完全被内源性启动子控制下表达的标记变体所取代。这些标签将通过荧光标记特异性纳米体可视化，或者，在EM的情况下，使用Ni-NTA纳米金或金标记纳米体。

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.