Defining the mechanism of actin-mediated spindle position sensing in mouse oocytes

最近的研究表明，细胞质肌动蛋白网络是动物生命开始时卵母细胞第一次减数分裂所必需的。这些肌动蛋白网络负责长距离运输染色体和减数分裂纺锤体的不对称定位。虽然有几条证据支持这样的观点，即可能需要肌动蛋白成核剂α和Spir来形成这些网络，但我们根本不了解它们在生理条件下的分子作用机制。因此，本建议的主要目的是了解如何Spir和cytosine成核复合物相互作用，在动物卵母细胞中的肌动蛋白丝成核，这些成核复合物是如何调节和肌动蛋白丝是如何组织成一个网络，以介导染色体/纺锤体定位。为了解决这些问题，我们将利用两个生物模型系统来研究减数分裂，即海星和小鼠，因为它们允许我们在动物卵母细胞中将遗传学、生物化学和先进的活细胞成像方法联合收割机结合起来。我们的方法有三个主要的互补和综合部分。首先，我们将使用定量活细胞成像测定来表征在可用的敲除小鼠模型中的P2P和Spir的功能。第二，我们将通过生化纯化和质谱鉴定海星中新的减数分裂辅助因子和调节因子。第三，我们将使用海星和小鼠卵母细胞在生理条件下表征和验证这些新发现的候选者。研究这两个系统将使我们能够有效地剖析卵母细胞特异性肌动蛋白网络的形成和功能的分子机制，并揭示其进化上保守的原则。阐明这些问题对于理解卵母细胞减数分裂的基本机制具有重要意义，同时也为研究肌动蛋白丝成核中保守的Spir-Spirt相互作用提供了第一个生理模型。这两个问题都是细胞生物学领域非常感兴趣的，对人类健康，特别是对了解不孕症有重要意义。

Recent work has revealed that cytoplasmic actin networks are required for the first meiotic division of oocytes at the beginning of animal life. These actin networks are responsible for transporting chromosomes over long distances and for asymmetric positioning of the meiotic spindle. Although several lines of evidence support the notion that the actin nucleators formin and Spir might be required to form these networks, we do not at all understand their molecular mechanism of action under physiological conditions. The principal aim of the present proposal is therefore to understand how Spir and formin nucleation complexes interact to nucleate actin filaments in animal oocytes, how these nucleation complexes are regulated and how the actin filaments are organized into a network to mediate chromosome/spindle positioning. To address these questions, we will take advantage of two biological model systems to study meiosis, namely starfish and mouse, because they allow us to combine genetics, biochemistry and advanced live cell imaging methods in animal oocytes. Our approach has three major complementary and integrated parts. First, we will use quantitative live cell imaging assays to characterize the function of formin and Spir in available knock out mouse models. Second, we will identify new meiotic formin/Spir accessory factors and regulators by biochemical purification and mass spectrometry in starfish. And third, we will characterize and validate these newly identified candidates under physiological conditions using both starfish and mouse oocytes. Studying the two systems will allow us to efficiently dissect the molecular mechanism underlying the formation and function of oocyte specific actin networks and reveal their evolutionarily conserved principles. Answering these questions will be of key importance for understanding the basic mechanisms of oocyte meiosis and at the same time provide one of the first physiological models to investigating the conserved Spir-formin interaction in actin filament nucleation. Both of these problems are of great interest to the field of cell biology and have important implications for human health, in particular for understanding infertility.