Project Summary/Abstract: Meiosis is the cell division process in which a diploid cell undergoes one round of DNA replication followed by two rounds of chromosome segregation to ultimately produce haploid gametes. Errors in meiotic chromosome segregation can result in miscarriage and trisomy conditions, such as Down syndrome. Therefore, studying meiotic chromosome segregation is important for understanding how errors in this process occur. The objective of this proposal is to determine the mechanisms that regulate proper chromosome segregation, focusing on unique events in meiosis I and meiosis II. These studies leverage the model organism S. cerevisiae, due to the ease of developing tools to address mechanistic questions. These innovative tools will allow the investigation of how cells establish microtubule-kinetochore attachments, how cells correct improper attachments, and how cells monitor the attachments through spindle checkpoint activity, which delays the cell cycle in the presence of unattached kinetochores. The rationale for the proposed research is that the questions focus on processes that are unique to meiosis but are likely to be highly conserved, allowing the findings in budding yeast to uncover general mechanisms of meiotic regulation. Strong preliminary data guide the following three specific aims: 1) determine how the number of crossovers and crossover position along the chromosome affects the establishment of correct kinetochore-microtubule attachments in meiosis I; 2) investigate how cells prevent persistent spindle checkpoint activity during meiosis; and, 3) determine how the phophoregulation of proteins at the meiotic kinetochore ensure proper kinetochore-microtubule attachments in meiosis II. In the first aim, strains have been developed to target crossovers at particular locations on a chromosome arm. Using time-lapse imaging, the strains will be monitored for the timing of establishing bioriented kinetochore microtubule attachments, and for the number of rounds of error correction of improper attachments. The second aim tests the novel hypothesis that cells have a developmentally programmed mechanism to overcome persistent spindle checkpoint activity in meiosis to ensure the production of gametes. The third aim analyzes how protein phosphatases counteract kinase activity to specifically ensure kinetochore assembly and the establishment of kinetochore-microtubule attachments specifically in meiosis II. The innovative approach of combining the latest imaging technologies to monitor kinetochore-microtubule attachments in engineered strains allows the testing of novel hypotheses about cell-cycle regulation. The proposed research is significant because the results are expected to reveal general principles of meiotic regulation important for proper chromosome segregation. Ultimately, the results will further our understanding of how errors in meiosis facilitate developmental abnormalities.