Role of Sas-4 in centrosome maturation

中心体由一对中心粒组成，周围围绕着中心粒物质(PCM)，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，这一过程被称为中心体成熟。虽然中心体成熟对于中心体的功能是必不可少的，但中心体成熟的机制仍不清楚。我们已经证明，果蝇SAS-4及其人类CPAP通过与PCM蛋白如CNN、Asterless(Asl)、Pericentin(PLP)和g-微管蛋白环复合体(G Turcs)相结合，在PCM向复制中心体的募集中发挥作用。然而，SAS-4是否以及如何在有丝分裂开始时触发PCM的初始募集还不完全清楚。在这项资助中，我们旨在剖析SAS-4在中心体成熟过程中的可能功能。由于SAS-4在细胞中是一种动态调节的蛋白质，我们将首先应用生化方法分析SAS-4中依赖于细胞周期的翻译后修饰，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招募过程中修饰的功能意义，我们将引入各自的点突变并产生转基因果蝇。在明确了SAS-4在体内有丝分裂PCM组装中的意义后，我们将从苍蝇胚胎提取液中纯化SAS-4介导的PCM，以在体外重建中心体的MTOC功能。拟议的实验将确定SAS-4在中心体成熟过程中是如何调节的，以及在体外功能重建中心体微管核活动所需的必要的PCM复合体。因此，本研究将为如何建立有丝分裂中心体以维持纺锤体的精确分裂提供新的见解。从上一个资助期(2014至2016)获得的结果我们获得了部分资金(GO2301/2-1)，这支持了一名博士生研究中心体生物发生的机制。在这一资助期的实验导致了两篇原创论文(郑Gooi等人，PNAS 2014和郑Ramani等人，《自然》杂志2016)1，2。在第一项工作中，我们证明了SAS-4在将SAS-4-PCM复合体与中心粒捆绑在一起的作用。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，这笔赠款部分帮助我们进行了纤毛和中心体相关实验，从而又产生了两部原创作品(Gabriel等人，EMBO J 20163和Mariappan等人，Mol Cell，正在审查中)。总体而言，这笔赠款极大地帮助我们建立了实验室，并进行了与中心体生物发生有关的实验。

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.