Homolog bi-orientation and segregation in oocyte acentrosomal meiosis

在第一次减数分裂过程中，同源染色体通过交叉连接，从相对的纺锤极（双向）附着到微管上，然后分离。在人类中，卵母细胞中染色体分离错误导致非整倍体，是流产、不孕和出生缺陷的主要原因。我们的长期目标是了解促进准确染色体分离的机制，以及使其易受染色体分离错误影响的卵母细胞纺锤体的特征。我们之前对雌性黑腹果蝇的研究已经建立了一个模型，在这个模型中，两种类型的微管附着被用于双向定向。横向附着物，即着丝点与微管的侧面相互作用，形成双向。然后是端上附着，即着丝点附着在微管末端，维持并分离双向同源物。果蝇卵母细胞的一个突出特征是中期I中央纺锤体，它作为“骨干”，在没有中心体的情况下将微管组织成双极结构。我们的工作表明，中央纺锤体在中期前的双向定位中起重要作用。对秀丽隐杆线虫和小鼠卵母细胞的研究表明，在无丝胞体减数分裂过程中，中期中央纺锤体可能是同源染色体双向定位所需的保守元件。在之前的资助期内，我们开发了几种工具来研究卵母细胞的双取向机制。这些工具包括抗RNAi转基因，以便制造关键蛋白质的种系特异性突变体。此外，我们有试剂，无论是转基因或抗体，以检测许多重要的蛋白质，调节染色体分离，包括着丝粒，着丝点，检查点和纺锤体蛋白。利用这些工具，我们将研究中心纺锤体与着丝点相互作用以促进双向定向的机制。端对端附着体的过早稳定很可能导致双向缺陷。因此，我们将研究侧向依恋和双向依恋的机制，以及如何调节向端上依恋的过渡。这些研究将集中于两个着丝点蛋白，CENP-C和SPC105R，它们需要加载其他几个着丝点和检查点蛋白。我们还将研究中央纺锤体如何与着丝点相互作用并促进准确的双向定位。这些研究将包括在果蝇中建立模型的实验，中心纺锤体突变会降低人类雌性的生育能力。这一建议的目的是通过一个目标来了解染色体分离的机制对卵母细胞的重要。在完成这项工作后，我们将深入了解着丝点如何调节从侧向和端上微管附着的转变。

During the first meiotic division, homologous chromosomes linked by chiasmata attach to microtubules from opposite spindle poles (bi-orientation) and then segregate. In humans, errors in chromosome segregation in the oocyte lead to aneuploidy and are the leading cause of miscarriage, infertility and birth defects. Our long-term goal is to understand the mechanisms that promote accurate chromosome segregation, and the features of the oocyte spindle that make it susceptible to chromosome segregation errors. Our previous research using Drosophila melanogaster females has led to a model in which two types of microtubule attachment are used for bi-orientation. Lateral attachments, where the kinetochores interact with the sides of microtubules, establish bi-orientation. Then end-on attachments, where the kinetochores attach to the end of microtubules, maintain and segregate bi-orientated homologs. A prominent feature of the Drosophila oocyte is the metaphase I central spindle, which functions as a “backbone”, organizing the microtubules into a bipolar structure in the absence of centrosomes. Our work has shown that the central spindle has an important role in bi-orientation during pro-metaphase. Studies in C. elegans and mouse oocytes indicate that the metaphase central spindle may be a conserved element required for the bi-orientation of homologous chromosomes during acentrosomal meiosis. In the previous funding period, we developed several tools to study the mechanisms of bi- orientation in oocytes. These tools include RNAi resistant transgenes in order to make germline-specific mutants of key proteins. Furthermore, we have the reagents, either transgenes or antibodies, to detect many of the important proteins that regulate chromosome segregation, including centromere, kinetochore, checkpoint and spindle proteins. With these tools, we will investigate the mechanisms by which the central spindle interacts with the kinetochores to promote bi-orientation. It is likely that premature stabilization of end-on attachments leads to bi-orientation defects. Therefore, we will investigate the mechanisms of lateral attachments and bi-orientation, and how the transition to end-on attachments is regulated. These studies will focus on two kinetochore proteins, CENP-C and SPC105R, which are required to load several other kinetochore and checkpoint proteins. We will also investigate how the central spindle interacts with the kinetochores and promotes accurate bi-orientation. These studies will include experiments to model in Drosophila, central spindle mutations that decrease fertility in human females. The Aims of this proposal are linked by a goal to understand the mechanisms of chromosome segregation important to oocytes. In completing this work, we will have gained insights into how kinetochores regulate the transition from lateral and end-on microtubule attachment.