Homolog bi-orientation and segregation in oocyte acentrosomal meiosis

在第一次减数分裂中，由交叉连接的同源染色体从相反的纺锤体极(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的错误会导致非整倍体，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，以及使其容易发生染色体分离错误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导致了一个模型，在这个模型中，两种类型的微管连接被用于双向。动点与微管两侧相互作用的侧向附着建立了双向。然后是末端附着体，其中动点连接到微管的末端，维持和分离双向同系物。果蝇卵母细胞的一个显著特征是中期I中心纺锤体，它的功能是“主干”，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，中心纺锤体在前中期的双向定向中起着重要的作用。对线虫和小鼠卵母细胞的研究表明，中期中心纺锤体可能是无着丝体减数分裂过程中同源染色体双向所必需的保守元件。在之前的资助阶段，我们开发了几种工具来研究卵母细胞双向定向的机制。这些工具包括抗RNAi的转基因，以制造关键蛋白质的种系特异性突变。此外，我们拥有转基因或抗体试剂，可以检测许多调节染色体分离的重要蛋白质，包括着丝粒、着丝粒、检查点和纺锤体蛋白质。利用这些工具，我们将研究中心主轴与动轴相互作用以促进双向的机制。端接附着体过早稳定可能会导致双向缺陷。因此，我们将研究侧向依恋和双向依恋的机制，以及如何调节向末端依恋的转变。这些研究将集中在两个动粒中心蛋白，CENP-C和SPC105R，这两个蛋白需要加载其他几个动粒和检查点蛋白。我们还将研究中心主轴如何与动轴相互作用，并促进准确的双向。这些研究将包括在果蝇身上建立模型的实验，果蝇的中心纺锤体突变会降低人类女性的生育力。这项提议的目的是为了了解染色体分离对卵母细胞重要的机制。在完成这项工作的过程中，我们将深入了解动点如何调节从侧向和端向微管附着的转变。

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.