Role of Sas-4 in centrosome maturation

Sas-4 在中心体成熟中的作用

• 批准号: 242472216
• 负责人: Professor Dr. Jay Gopalakrishnan, Ph.D.
• 金额: --
• 依托单位: Labor für Zentrosomen- und Zytoskelettbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/242472216?language=en
• 关键词: Role; Sas; centrosome; maturation

Centrosomes consist of a pair of centrioles surrounded by Peri-Centriolar Material (PCM) acting as the major microtubule-organizing centers (MTOC) in animal cells. In order for a centrosome to nucleate microtubules at the onset of mitosis, it has to recruit PCM proteins, a process termed as centrosome maturation. Although centrosome maturation is essential for the functionality of centrosomes, the mechanisms of centrosome maturation remain unclear. Others and we have shown that Drosophila Sas-4 and its human counterpart CPAP plays a role in PCM recruitment to duplicated centrosomes by associating with PCM proteins such as Cnn, Asterless (Asl), Pericentrin (PLP) and components of g-tubulin ring complexes (g TuRCs). However, whether and how Sas-4 plays a role in triggering initial PCM recruitment at the onset of mitosis is not completely understood. In this grant, we aim to dissect the possible function of Sas-4 in centrosome maturation. Since, Sas-4 is a dynamically regulated protein in cells, first, we will apply biochemical methods to analyze the cell cycle dependent posttranslational modifications in Sas-4 and identify its phosphorylation status at the onset of mitosis. To dissect the functional significance of modifications during mitotic PCM recruitment, we will introduce respective point mutations and generate transgenic Drosophila. Upon identifying the significance of Sas-4 in mitotic PCM assembly in vivo, we will purify Sas-4-mediated PCM from fly embryonic extracts to functionally reconstitute MTOC functions of centrosomes in vitro. The proposed experiments will identify how Sas-4 is regulated during centrosome maturation and the essential PCM complexes required to functionally reconstituting microtubule-nucleating activity of a centrosome in vitro. Thus, the current study will provide new insights into how mitotic centrosomes are built to maintain spindle poles for accurate cell division.Results obtained from the previous funding period (2014 to 2016) We were granted a partial funding (GO2301/2-1), which supported a doctoral student to study the mechanisms of centrosome biogenesis. Experiments from this funding period resulted in two original publications (Zheng and Gooi et al., PNAS 2014 and Zheng and Ramani et al., Nature Commun 2016)1, 2. In the first work, we demonstrated that Sas-4 plays a role in tethering Sas-4-PCM complexes to a centriole. In the second work, we showed the significance of Sas-4-tubulin interaction in centriole and primary cilium length control. In addition, this grant partially helped us in performing cilia and centrosome-related experiments, which resulted in two more original works (Gabriel et al., EMBO J 20163 and Mariappan et al., Mol Cell, Under review). Overall, this grant greatly helped in setting up our laboratory and pursuing experiments related to centrosome biogenesis.

中心体由一对中心粒组成，中心粒被中心粒周围物质（PCM）包围，是动物细胞中主要的微管组织中心（MTOC）。为了使中心体在有丝分裂开始时形成微管，它必须招募PCM蛋白，这一过程称为中心体成熟。虽然中心体成熟对中心体的功能至关重要，但中心体成熟的机制尚不清楚。其他人和我们已经表明，果蝇Sas-4及其人类对应体CPAP通过与PCM蛋白（如Cnn、Asterless （Asl）、Pericentrin （PLP）和g-微管蛋白环复合物（g TuRCs）的组分）相关，在PCM募集到复制中心体中发挥作用。然而，Sas-4是否以及如何在有丝分裂开始时触发初始PCM募集中发挥作用尚不完全清楚。在这项资助中，我们的目标是剖析Sas-4在中心体成熟中的可能功能。由于Sas-4在细胞中是一种动态调节的蛋白，首先，我们将应用生化方法分析Sas-4的细胞周期依赖的翻译后修饰，并确定其在有丝分裂开始时的磷酸化状态。为了剖析有丝分裂PCM募集过程中修饰的功能意义，我们将引入相应的点突变并产生转基因果蝇。在确定了Sas-4在体内有丝分裂PCM组装中的重要性之后，我们将从果蝇胚胎提取物中纯化Sas-4介导的PCM，在体外功能上重建中心体的MTOC功能。本实验将确定在中心体成熟过程中Sas-4是如何被调控的，以及在体外重构中心体微管成核活性所需的基本PCM复合物。因此，目前的研究将为有丝分裂中心体如何构建以维持纺锤杆以进行准确的细胞分裂提供新的见解。上一期资助（2014 - 2016）获得部分资助（GO2301/2-1），支持博士生研究中心体生物发生机制。该资助期的实验产生了两篇原创论文（Zheng and Gooi et al., PNAS 2014; Zheng and Ramani et al., Nature comm 2016） 1,2。在第一项工作中，我们证明了Sas-4在将Sas-4- pcm复合物系在中心粒上发挥作用。在第二项工作中，我们展示了sas -4-微管蛋白相互作用在中心粒和初级纤毛长度控制中的意义。此外，这笔资助部分帮助我们进行了纤毛和中心体相关的实验，从而产生了两篇原创作品（Gabriel et al.， EMBO J 20163和Mariappan et al., Mol Cell, Under review）。总的来说，这笔拨款对我们建立实验室和开展中心体生物发生相关的实验有很大的帮助。