Homolog orientation and segregation in acentrosomal meiosis

描述(申请人提供)：在第一次减数分裂中，由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。这一过程中的缺陷会导致受精卵中的非整倍体，通常会导致 发育中的胚胎。在人类中，非整倍体是导致女性自然流产和不孕的主要原因，并导致唐氏综合症、特纳综合征或克莱菲尔特综合征等疾病。在包括哺乳动物和昆虫在内的许多生物中，卵母细胞减数分裂纺锤体缺乏中心体。在有丝分裂纺锤体极点没有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。在有中心体的细胞中，在极点和动点之间形成的微管连接有助于姐妹染色体(有丝分裂)或同源染色体(减数分裂I)的双向定向。在无着丝体细胞中，新的机制可能被用来双向定位同系物。我们发现，包括染色体乘客复合体(CPC)在内的一组中心纺锤体蛋白对双极纺锤体的形成和同系物的定位至关重要。这些蛋白质招募并组织在纺锤体中心重叠的反平行微管。这些微管如何调节染色体行为尚不清楚。在这个方案中，我们将研究果蝇卵母细胞无着丝体纺锤体中同源定位的机制。即使在没有微管的情况下，CPC也会被招募到染色体上，但与有丝分裂细胞不同的是，在着丝粒上没有发现CPC蛋白。相反，CPC被发现在染色体周围的环中，它在那里招募调节纺锤体组装的因子，如Subito。环结构还提供了在没有中心体的情况下引导纺锤体两极的机制。为了研究中心纺锤体在同源定位中的作用，我们将使用荧光标记的蛋白质和活体成像来研究相对于纺锤体组装和中心纺锤体的建立的同源定位的时间。我们还将确定着丝点在染色体比对和分离中的作用。由于这些基因是必不可少的，我们将使用在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，以测试不同动粒成分的作用。最后，我们将检验这一假设，即同源基因的双向依赖于染色体相关微管和中心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.