Regulation and cellular functions of the Arp2/3 nucleation complex in actin-driven chromosome transport in oocyte meiosis

卵母细胞减数分裂是一种高度专业化的细胞分裂形式，产生受精卵，因此是所有动物有性繁殖所必需的，与人类健康高度相关。事实上，卵母细胞减数分裂错误导致的非整倍体是人类妊娠丢失和出生缺陷的主要原因，这在西方国家随着母亲年龄的增加而发展成为一个主要的社会问题。卵母细胞减数分裂是专门化的，因为卵母细胞非常大，为了保存积累的营养以支持早期胚胎发育，卵母细胞以极不对称的方式分裂。最近在各种动物模型物种的研究中发现，肌动蛋白细胞骨架具有重要的、保守的功能，即在极大的卵母细胞胞浆中运输和定位染色体--这种功能是由小体细胞中的微管介导的。最近在小鼠卵母细胞和非洲爪哇卵提取液中的研究以及我们在海星卵母细胞中的初步数据表明，Arp2/3复合体在这些过程中具有重要和保守的作用。正如其他人在小鼠卵母细胞中所显示的那样，以及我们在海星卵母细胞中的初步数据表明，在这种细胞环境中，Arp2/3复合体是由染色体上本地产生的RanGTP招募并激活的。这是第一个已知的将肌动蛋白成核与染色质联系起来的机制，因此很可能是肌动蛋白驱动的染色体在卵母细胞中运输和定位的关键。RanGTP是如何招募和激活Arp2/3复合体的，目前还不清楚。在优先计划SPP1464的第一个资助期，我们建立了一个海星卵母细胞作为一个实验系统，用于纯化F-肌动蛋白调节蛋白以及生化和细胞特性。在这个项目中，我们将在这个实验系统的基础上纯化和鉴定参与RanGTP介导的Arp2/3激活的分子。为此，我们将结合已经开发的鉴定RanGTP激活的微管调节因子的方法与F-肌动蛋白相互作用的纯化。我们将在海星卵母细胞的活细胞分析中验证这些新确定的候选对象，并进一步表征它们的细胞功能。同时，我们将在体外成核分析中对这些新发现的Arp2/3调控因子进行生化表征。人们普遍认为RanGTP在组织染色体周围的微管纺锤体方面具有关键功能。该项目的主要目的是在分子和细胞水平上确定RanGTP在调节Arp2/3复合体使染色质上的肌动蛋白成核，从而推动染色体在大卵母细胞中的运输和定位方面具有同样重要的功能，这是防止卵子非整倍体的关键机制。

Oocyte meiosis is a highly specialized form of cell division that produces the fertilizable egg, and is therefore essential for sexual reproduction of all animal species and is highly relevant to human health. Indeed, aneuploidy resulting from errors in oocyte meiosis is the leading cause of pregnancy loss and birth defects in humans that is developing into a major social issue with increasing maternal age in Western countries.Oocyte meiosis is specialized, because oocytes are very large and divide with extreme asymmetry in order to preserve accumulated nutrients to support early embryonic development. As it recently emerged from studies in various animal model species, the actin cytoskeleton has major, conserved functions in transporting and positioning chromosomes in the exceptionally large cytoplasm of oocytes – functions that are mediated by microtubules in small somatic cells. Recent studies in mouse oocytes and Xenopus egg extracts, as well as our preliminary data in starfish oocytes showed that the Arp2/3 complex has important and conserved roles in these processes. As shown in mouse oocytes by others and as indicated by our preliminary data in starfish oocytes, in this cellular context the Arp2/3 complex is recruited and activated by RanGTP produced locally on chromosomes. This is the first known mechanism that links actin nucleation to chromatin, and is therefore likely to be key to actin-driven transport and positioning of chromosomes in oocytes. How RanGTP recruits and activates the Arp2/3 complex is not at all understood.In the course of the first funding period of the Priority Programme SPP 1464 we established starfish oocytes as an experimental system for purification and biochemical and cellular characterization of F-actin regulators. In this project we will build on this experimental system to purify and identify the molecules involved in RanGTP-mediated activation of Arp2/3. To this end, we will combine assays that had been developed to identify microtubule regulators activated by RanGTP with purification of F-actin interactors. We will validate these newly identified candidates in live cell assays in starfish oocytes and further characterize their cellular functions. Simultaneously, we will biochemically characterize these newly identified regulators of Arp2/3 in in vitro nucleation assays.It is widely accepted that RanGTP has key functions in organizing the microtubule spindle around chromosomes. The major aim of the proposed project is to establish at the molecular and cellular level that RanGTP has equally important functions in regulating the Arp2/3 complex to nucleate actin on chromatin and thereby driving chromosome transport and positioning in large oocytes, a mechanism essential to prevent aneuploidy of eggs.