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，因此对所有动物物种的有性生殖至关重要，与人类健康高度相关。事实上，在西方国家，由于卵母细胞减数分裂错误而导致的非整倍体是导致人类流产和出生缺陷的主要原因，随着母亲年龄的增长，这正在发展成为一个重大的社会问题。卵母细胞减数分裂是专门的，因为卵母细胞非常大，并且以极端不对称的方式分裂，以保存积累的营养物质，以支持早期胚胎发育。正如最近从各种动物模型物种的研究中发现的那样，肌动蛋白细胞骨架在卵母细胞异常大的细胞质中运输和定位染色体方面具有主要的保守功能-这些功能由小型体细胞中的微管介导。最近在小鼠卵母细胞和爪蟾卵提取物中的研究，以及我们在海星卵母细胞中的初步数据表明Arp 2/3复合物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示，以及我们在海星卵母细胞中的初步数据所示，在这种细胞背景下，Arp 2/3复合物被染色体上局部产生的RanGTP募集和激活。这是第一个已知的连接肌动蛋白成核和染色质的机制，因此可能是肌动蛋白驱动的运输和定位的染色体在卵母细胞中的关键。RanGTP如何招募和激活Arp 2/3复合体还不完全清楚。在优先计划SPP 1464的第一个资助期内，我们建立了海星卵母细胞作为F-肌动蛋白调节剂纯化和生化及细胞表征的实验系统。在这个项目中，我们将建立在这个实验系统，以纯化和鉴定参与RanGTP介导的Arp 2/3激活的分子。为此，我们将联合收割机的分析，已开发出识别微管调节激活的RanGTP与纯化的F-肌动蛋白相互作用。我们将在海星卵母细胞的活细胞测定中验证这些新鉴定的候选物，并进一步表征它们的细胞功能。同时，我们将在体外成核试验中对这些新鉴定的Arp 2/3调节剂进行生物化学表征。该项目的主要目的是在分子和细胞水平上确定RanGTP在调节Arp 2/3复合物以使染色质上的肌动蛋白成核，从而驱动大卵母细胞中的染色体运输和定位方面具有同等重要的功能，这是防止卵细胞非整倍性所必需的机制。

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.