DBT-DFG Indo-German Research Proposal: The recruitment of the γ-TuRC to the Sas-6 cartwheel for centriole microtubule assembly

DBT-DFG 印度-德国研究提案：将 γ-TuRC 募集到 Sas-6 侧手翻以进行中心粒微管组装

• 批准号: 465380792
• 负责人: Professor Dr. Elmar Schiebel
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/465380792?language=en
• 关键词: DBT; DFG; Indo; German; Research

Centrioles are microtubule-based subcellular structures that constitute the core of centrosomes, the main microtubule-nucleating organelles in animal cells. In G1 phase of the cell cycle two centrioles surrounded by the pericentriolar material give rise to a functional centrosome. During S phase, each of the two mother centrioles initiates assembly of a new daughter centriole at its proximal end. The orthogonally arranged mother-daughter centriole pair stays connected during mitosis when each pair associates with one of the two poles of the mitotic spindle. With cytokinesis each cell inherits one centriole pair that then disengages. Centrioles consist of 9 microtubule triples that each contain radially arranged A, B and C microtubules. The initial assembly of centriolar microtubules requires a cartwheel-like nine-fold symmetric scaffold consisting of the protein Sas-6 that provides the base for assembly of the A-microtubules. A-microtubule assembly is a crucial step for the duplication of centrioles, the biogenesis of centrosomes and the correct formation of the mitotic spindle and its failure has been linked to chromosome missegregation and cancer. Therefore, it is of fundamental importance to understand how centrioles and in particular the A-tubules, as a key event in early centriole biogenesis, assemble mechanistically. Cryo-electron microscopy (cryo-EM) of human procentrioles previously indicated presence of cap-like structure at the base of the centriolar microtubule resembling the γ-tubulin ring complex (γ-TuRC), the major microtubule-nucleating entity. Recently, Manna has shown interaction of the γ-TuRC with SAS-6 and Schiebel reported the structure of the γ-TuRC based on cryo-EM analysis. Although, these studies indicate a molecular link of the cartwheel with the centriolar microtubules, how the γ-TuRCs are recruited, organized and activated for assembly of the A-microtubules are not understood and will be addressed in this collaborative study as part of DBT-DFG between India and Germany. Our working model is that the Sas-6 cartwheel recruits the γ-TuRC in S phase through its C-terminus with the subsequent activation of the γ-TuRC and the nucleation of the A-tubules followed by the assembly of the B- and C-tubules. To decipher A-tubule formation in greater detail, we will study the interaction of Sas-6 with the γ-TuRC using negative stain EM and cross-link mass spectrometry analysis (2.3.1), the role of PLK4-mediated phosphorylation in γ-TuRC-C-Sas-6 interaction (2.3.2), cryo-EM analysis of the γ-TuRC-Sas-6 complex (2.3.3), how events at the γ-TuRC-Sas-6 super-complex lead to A-tubule formation using super resolution microscopy (2.3.4) and biochemical approaches (2.3.5). Altogether, our proposal will unravel the mechanisms of centriolar microtubule assembly from a novel perspective, by combining ultra-structural analyses of the key protein complexes with regulation of their interactions that lead to the biogenesis of centrioles.

中心粒是以微管为基础的亚细胞结构，构成中心体的核心，中心体是动物细胞中主要的微管成核细胞器。在细胞周期的G1期，两个中心粒被中心粒周围的物质包围，形成一个功能中心体。在S期，两个母亲中心粒中的每一个在其近端开始组装一个新的女儿中心粒。在有丝分裂过程中，当每对中心粒与有丝分裂纺锤体的两极之一时，正交排列的母子中心粒对保持连接。随着胞质分裂，每个细胞继承一对中心粒，然后分离。中心粒由9个微管三联体组成，每个微管三联体含有放射状排列的A、B和C微管。中心粒微管的初始组装需要一个侧手翻状的九重对称支架，该支架由蛋白Sas-6组成，为A-微管的组装提供基础。微管组装是中心粒复制、中心体发生和纺锤体正确形成的关键步骤，其失败与染色体错误分离和癌症有关。因此，了解中心粒，特别是A-小管，作为早期中心粒生物发生的关键事件，如何组装机械是至关重要的。人原中心粒的冷冻电子显微镜（cryo-EM）先前表明中心粒微管基部存在帽状结构，类似于γ-微管蛋白环复合物（γ-TuRC），这是主要的微管成核实体。最近，Manna已经显示了γ-TuRC与SAS-6的相互作用，Schiebel基于冷冻-EM分析报道了γ-TuRC的结构。尽管这些研究表明侧手翻与中心粒微管存在分子联系，但γ-TuRC如何被招募、组织和激活以组装A-微管尚不清楚，将在本合作研究中作为印度和德国之间DBT-DFG的一部分进行讨论。我们的工作模型是，Sas-6侧手翻通过其C-末端招募S期的γ-TuRC，随后激活γ-TuRC并使A-小管成核，然后组装B-和C-小管。为了更详细地解释A-小管的形成，我们将使用负染EM和交联质谱分析来研究Sas-6与γ-TuRC的相互作用（2.3.1），PLK 4介导的磷酸化在γ-TuRC-C-Sas-6相互作用中的作用（2.3.2），γ-TuRC-Sas-6复合物的冷冻EM分析（2.3.3），使用超分辨率显微镜（2.3.4）和生物化学方法（2.3.5）研究γ-TuRC-Sas-6超复合物中的事件如何导致A-小管形成。总而言之，我们的建议将从一个新的角度解开中心粒微管组装的机制，通过结合超微结构分析的关键蛋白质复合物与调节它们的相互作用，导致中心粒的生物起源。