Homolog orientation and segregation in acentrosomal meiosis

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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.