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

中心粒是基于微管的亚细胞结构，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物质包围，形成一个功能中心体。在S期，两个母中心粒中的每一个在其近端启动一个新的子中心粒的组装。在有丝分裂过程中，当母女中心粒对与有丝分裂纺锤体的两极中的一个结合时，母女中心粒对保持连接。在细胞分裂过程中，每个细胞继承一对中心粒，然后分离。中心粒由9个微管组组成，每个微管组含有径向排列的A、B和C微管。中心微管的初始组装需要一个由蛋白质Sas-6组成的车轮状九重对称支架，该支架为a微管的组装提供了基础。微管组装是中心粒复制、中心体生物发生和有丝分裂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，特别是作为早期中心粒生物发生的关键事件的a小管如何机械地组装具有重要意义。人类前心粒的冷冻电镜（cryo-EM）先前显示，在中心微管的底部存在类似于微管成核实体γ-微管蛋白环复合物（γ-TuRC）的帽状结构。最近，甘露中γ-TuRC与SAS-6相互作用，Schiebel报道了基于低温电镜分析的γ-TuRC结构。尽管这些研究表明了侧车轮与向心微管之间的分子联系，但γ- turc是如何被招募、组织和激活以组装a微管的尚不清楚，这将在印度和德国之间的DBT-DFG合作研究中得到解决。我们的工作模型是，Sas-6侧轮通过其c端在S期招募γ-TuRC，随后γ-TuRC被激活，a小管成核，然后是B小管和c小管的组装。为了更详细地解释a小管的形成，我们将使用负染色EM和交联质谱分析（2.3.1）研究Sas-6与γ-TuRC的相互作用，plk4介导的磷酸化在γ-TuRC- c -Sas-6相互作用中的作用（2.3.2），对γ-TuRC-Sas-6复合物进行低温电镜分析（2.3.3），使用超分辨率显微镜（2.3.4）和生化方法（2.3.5）研究γ-TuRC-Sas-6超复合物的事件如何导致a小管的形成。总之，我们的建议将通过结合关键蛋白质复合物的超结构分析和导致中心粒生物发生的相互作用的调节，从一个新的角度揭示中心粒微管组装的机制。

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.