The oocyte's progression through meiosis: Involvement of a heart disease-associated protein

总结 成功的胚胎发育依赖于雌配子通过减数分裂正确地进行。 减数分裂纺锤体的组装和定位是这一过程的关键部分，基因敲除， 损害这些过程导致女性不育。卵母细胞纺锤体的组织和定位是精心安排的 肌动蛋白，涉及肌动蛋白相关蛋白质在细胞质网络和卵母细胞皮层。我们 对卵母细胞中肌动蛋白相关蛋白的研究已经确定nexilin参与了这些事件， 显示RNAi介导的nexilin敲低导致减数分裂停滞和异常的 卵母细胞肌动蛋白的组织。我们也有证据表明nexilin的缺失会影响肌动蛋白调节途径 涉及含LIM结构域的激酶（LIMK）及其底物，肌动蛋白解聚因子 cofilin LIMK-切丝蛋白途径影响F-肌动蛋白丝解聚成单体G-肌动蛋白， 因此，这是nexilin影响肌动蛋白依赖性过程的有希望的机制。Nexilin是 由于其在扩张性和肥厚性心肌病（DCM和HCM， 分别）。因此，这里提出的研究的影响是广泛的，与生殖有关， 卵母细胞生物学、肌肉功能和心肌病。扩张型心肌病的发病通常在40 - 60岁之间， 我们假设NEXN基因中的功能破坏突变可能是女性不育的原因之一 在生育期，然后在以后的生活中患心脏病。鉴于对nexilin知之甚少， 总体目标是阐明nexilin的功能，它与LIMK-cofilin通路的联系，以及nexilin 功能障碍导致哺乳动物卵母细胞的异常。我们将通过以下方式实现这些目标： 具体目标。在目标1中，我们将建立在我们的数据从RNAi介导的敲低nexilin在卵母细胞， 建立卵母细胞特异性nexilin条件性敲除（cKO）模型，分析nexilin缺失的影响 在卵母细胞中的活性，在体内和体外。目标2将使用最先进的研究在细胞力学，活细胞 成像和定量分析来阐明纺锤体组织缺陷的机制 以及与nexilin缺乏相关的易位。这一目标将检验异常纺锤体 与nexilin或LIMK-cofilin途径缺陷相关的定位归因于（a）异常的 用于将纺锤体拉向卵母细胞外周的皮质锚定的张力，或（B）基于肌动蛋白的 纺锤体在卵母细胞质中的运动。目标3将研究nexilin的突变形式如何影响 卵母细胞、卵子和早期胚胎。这项工作将是一个宝贵的评估的严重性， 疾病相关的形式，并提供答案的问题，如果一个女人有一个这些NEXN 突变对她的卵母细胞会有什么影响总的来说，这个项目将揭示一个糟糕的 通过阐明nexilin在卵母细胞和一般情况下的功能，了解但重要的健康相关蛋白。在 反过来，这项工作将转化为理解心肌细胞和其他细胞类型中的nexilin功能。

SUMMARY Successful embryonic development is dependent on the female gamete progressing correctly through meiosis. Assembly and positioning of the meiotic spindle is a crucial part of this process, with gene knockouts that impair these processes causing female infertility. Oocyte spindle organization and positioning is orchestrated by actin, involving actin-associated proteins in a cytoplasmic meshwork and in the oocyte cortex. Our research on actin-associated proteins in oocytes has identified nexilin as involved in these events, with data presented here showing that RNAi-mediated knockdown of nexilin results in meiotic arrest and aberrant organization of oocyte actin. We also have evidence that loss of nexilin affects the actin regulatory pathway involving the LIM-domain containing kinase (LIMK) and its substrate, the actin-depolymerization factor cofilin. The LIMK-cofilin pathway affects the depolymerization of F-actin filaments to monomeric G-actin, and thus this is a promising mechanism by which nexilin could impact actin-dependent processes. Nexilin is of broader relevance as well, due to its role in dilated and hypertropic cardiomyopathies (DCM and HCM, respectively). Thus, the impact of the research proposed here is wide-ranging, with relevance to reproduction, oocyte biology, muscle function, and cardiomyopathies. With onset of DCM typically being in one's 40s-60s, we hypothesize that a function-disrupting mutation in the NEXN gene could be a cause of female infertility during reproductive years, and then of heart disease later in life. Given that little is known about nexilin, our overall goal is to elucidate the functions of nexilin, its connection to the LIMK-cofilin pathway, and how nexilin dysfunction contributes to abnormalities in mammalian oocytes. We will achieve these goals with following Specific Aims. In Aim 1, we will build on our data from RNAi-mediated knockdown nexilin in oocytes, and develop an oocyte-specific nexilin conditional knockout (cKO) model, to analyze the effects of loss of nexilin activity in oocytes, in vivo and in vitro. Aim 2 will use state-of-the-art studies in cellular mechanics, live-cell imaging, and quantitative analyses to elucidate the mechanisms underlying the defects in spindle organization and translocation associated with nexilin deficiency. This aim will test the hypotheses that aberrant spindle positioning associated with deficiencies in nexilin or the LIMK-cofilin pathway are attributed to (a) aberrant tension for cortical anchoring for spindle pulling to the oocyte periphery, or (b) defects in actin-based movement of the spindle in the oocyte cytoplasm. Aim 3 will investigate how mutated forms of nexilin affect oocytes, eggs, and early embryos. This work will be an invaluable assessment of the severity of different disease-associated forms, and also provide answers to the question of if a woman has one of these NEXN mutations, what would the effects be on her oocytes? Overall, this project will shed light on a poorly understood but significant health-relevant protein by elucidating nexilin functions in oocytes and in general. In turn, this work will translate to understanding nexilin functions in cardiomyocytes and other cell types.