The role of the centriolar CEP350 network in centrobin removal from centrioles and distal appendage/CEP19 recruitment

中心体对细胞的组织和分裂至关重要，其结构和功能的缺陷与各种人类疾病有关。这项建议侧重于中心体蛋白CEP350，它对控制CEP19的中心粒长度、稳定性、组装亚远端(SDA)和远端(DA)附件以及中心粒募集至关重要。DA组装和CEP19募集对纤毛发生是必不可少的，它们组装的缺陷与各种疾病有关，如纤毛疾病和病态肥胖(CEP19)。CEP350还调节中心粒蛋白Centrobin，它是中心体正常功能所必需的，并通过蛋白质分解从成熟的中心粒中特异性地移除。中心酶去除的失调与多发性纳米症和癌症有关。尽管CEP350对DA的组装、CEP19的募集和中心蛋白的去除具有重要作用，但它的分子功能却鲜为人知。了解CEP350如何调控这些过程对于阐明中心体生物学和疾病机制中的关键开放问题至关重要，因此是本提案的重点。根据我们的初步发现，我们认为CEP350是一种多功能蛋白，它与DISCO网络组件(几种中心粒蛋白与CEP350一起进行DA组装)、DA蛋白和CEP19协同完成其功能。我们的研究结合了教授实验室的专业知识。Pereira和Schiebel在中心粒生物学、CEP350分析、DA组装和纤毛发生方面，主要关注两个目标。首先，我们旨在探索CEP350、SDAS、E3连接酶TRIM37和我们新发现的激酶CRM5从中心粒上去除中心知母蛋白的详细机制。此外，我们还将调查中央知母蛋白的去除是否对DA组装是必要的。其次，我们将研究CEP350相互作用的蛋白质和母中心粒特异性修饰如何调节DA组装和CEP19中心粒在成熟中心粒的招募。为了实现这些目标，我们将使用各种技术，包括基因敲除、信号强度测量、超分辨/扩展显微镜、电子显微镜、Mflux纳米显微镜和对CEP350相互作用蛋白质子集的磷酸化位点的质谱分析。通过揭示CEP350是如何控制中心体和DA组装途径的，我们的研究将有助于回答中心体生物学、纤毛组装以及缺陷如何导致疾病的关键问题。

The centrosome is vital for cellular organization and division, and defects in its structure and function are linked to various human diseases. This proposal focuses on the centrosome protein CEP350 that is critical for controlling centriole length, stability, assembling subdistal (SDA) and distal (DA) appendages and centriole recruitment of CEP19. DA assembly and CEP19 recruitment are essential for ciliogenesis, and defects in their assembly are linked to various diseases such as ciliopathies and morbid obesity (CEP19). CEP350 also regulates the centriolar protein centrobin, which is necessary for proper centrosome function and is specifically removed from maturing centrioles by proteolysis. Dysregulation of centrobin removal is linked to Mulibrey nanism and cancer. Despite the importance of CEP350 for DA assembly, CEP19 recruitment and centrobin removal, its molecular functions are poorly understood. Understanding how CEP350 regulates these processes is critical for elucidating key open questions in centrosome biology and disease mechanisms and therefore stands in the focus of this proposal. Based on our preliminary findings, we propose that CEP350 is a multifunctional protein that cooperates with DISCO network components (several centriolar proteins needed together with CEP350 for DA assembly), DA proteins and CEP19 in order to achieve its functions. Our study, which combines the expertise of the laboratories of Profs. Pereira and Schiebel in centriole biology, CEP350 analysis, DA assembly and ciliogenesis, focuses on two major goals. Firstly, we aim to explore the detailed mechanisms of centrobin removal from centrioles by CEP350, SDAs, the E3 ligase TRIM37 and the by us newly identified kinase CRM5. In addition, we will also investigate whether centrobin removal is essential for DA assembly. Secondly, we will investigate how CEP350 interacting proteins and mother centriole specific modifications regulate DA assembly and CEP19 centriole recruitment at maturing centrioles. To achieve these goals, we will use various techniques, including gene knockouts, signal intensity measurements, super-resolution/expansion microscopy, electron microscopy, Minflux nanoscopy and mass spectrometry analysis of phosphorylation sites in a subset of CEP350 interacting proteins. By uncovering how CEP350 controls centrobin and DA assembly pathways, our study will help answering critical questions in centrosome biology, cilia assembly and how defects lead to disease.