Homolog orientation and segregation in acentrosomal meiosis

描述（由申请人提供）：在第一次减数分裂期间，通过交叉连接的同源染色体与纺锤体微管相互作用并分离到相反的两极。这一过程中的缺陷导致受精卵的非整倍体，通常导致受精卵的死亡。 胚胎发育在人类中，非整倍体是女性自然流产和不孕不育的主要原因，并导致唐氏综合症、特纳综合症或克氏综合症等疾病。在许多生物中，包括哺乳动物和昆虫，卵母细胞减数分裂纺锤体缺乏中心体。在有丝分裂纺锤体两极缺乏微管组织中心的情况下，染色体产生刺激纺锤体组装的信号。在具有中心体的细胞中，极和动粒之间形成的微管连接促进姐妹篇（有丝分裂）或同源染色体（减数分裂I）的双向定向。在acentrosomal细胞中，新的机制可能会被用来双向同源。我们已经发现，一组中央纺锤体蛋白，包括染色体乘客复合物（CPC）是至关重要的双极纺锤体的形成和同源物的方向。这些蛋白质募集并组织在纺锤体中心重叠的反平行微管。这些微管如何介导染色体行为尚不清楚。 在本研究中，我们将探讨果蝇卵母细胞纺锤体中同源物的定位机制。即使在没有微管的情况下，CPC也被招募到染色体上，但与有丝分裂细胞不同，CPC蛋白不在着丝粒上。相反，CPC被发现在染色体周围的环中，在那里它招募调节纺锤体组装的因子，如Subito。环结构也提供了一种机制，指导纺锤体双极性的中心体的情况下。为了研究中心纺锤体在同源物取向中的作用，我们将使用荧光标记的蛋白质和活体成像来研究同源物取向相对于纺锤体组装和中心纺锤体建立的时间。我们还将确定着丝粒在染色体排列和分离中的作用。由于这些基因是必需的，我们将使用果蝇中可用的复杂遗传工具来产生缺乏这些蛋白质的卵母细胞。这包括新开发的生殖系RNAi和生殖系克隆，以测试不同动粒组分的作用。最后，我们将测试的假设，同源物的双向取向依赖于染色体相关和中央纺锤体微管之间的相互作用。

DESCRIPTION (provided by applicant): During the first meiotic division, homologous chromosomes linked by chiasmata interact with spindle microtubules and segregate to opposite poles. Defects in this process lead to aneuploidy in the fertilized egg and usually in death of the developing embryo. In humans, aneuploidy is a leading cause of spontaneous abortions and infertility in women and causes diseases such as Down, Turner or Klinefelter syndromes. In many organisms, including mammals and insects, the oocyte meiotic spindle lacks centrosomes. In the absence of the microtubule-organizing center found at mitotic spindle poles, the chromosomes generate a signal which stimulates spindle assembly. In cells with centrosomes, the microtubule connections formed between the poles and the kinetochores facilitates bi-orientation of sisters (mitosis) or homologous chromosomes (meiosis I). In acentrosomal cells, novel mechanisms may be employed to bi-orient the homologs. We have found that a group of central spindle proteins, including the Chromosome Passenger Complex (CPC) is critical for formation of a bipolar spindle and orientation of the homologs. These proteins recruit and organize the antiparallel microtubules overlap in the center of the spindle. How these microtubules mediate chromosome behavior is not known. In this proposal, we will investigate the mechanisms of homolog orientation in the acentrosomal spindle of Drosophila oocytes. The CPC is recruited to chromosomes even in the absence of microtubules but unlike mitotic cells, CPC proteins are not found at the centromeres. Instead, the CPC is found in a ring around the chromosomes where it recruits factors which regulate spindle assembly such as Subito. The ring structure also provides a mechanism for directing spindle bipolarity in the absence of centrosomes. To investigate the role of the central spindle in homolog orientation, we will use fluorescently tagged proteins and live imaging to investigate the timing of homolog orientation relative to spindle assembly and establishment of the central spindle. We will also determine the role of kinetochores in chromosome alignment and segregation. Since these genes are essential, we will use sophisticated genetic tools available in Drosophila to generate oocytes lacking these proteins. This includes newly developed germ line RNAi and germ line clones to test the role of different kinetochore components. Finally, we will test the hypothesis that the bi-orientation of homologs depends on an interaction between chromosome associated and central spindle microtubules.