The seemingly straightforward function of the centromere in directing chromosome segregation is difficult to reconcile with multiple complexities of the underlying molecular machinery, particularly rapid evolution of both centromere DNA and proteins and seemingly redundant pathways linking the DNA to spindle microtubules. This project focuses on centromere drive as a key to unlocking centromere complexity. Selfish centromere DNA sequences bias their transmission to the egg in female meiosis, while centromere proteins evolve to suppress fitness costs of drive while maintaining essential centromere functions. Our recent work determined how selfish centromeres interact with spindle microtubules to bias their segregation. We developed mouse model systems exploiting natural variation in mouse centromere DNA, defined tubulin detyrosination as the key post-translational modification creating meiotic spindle asymmetry, showed that microtubule-destabilizing proteins act as drive effectors exploited by selfish centromeres, established an integrated model for both drive and suppression, and sequenced Murinae genomes for molecular evolution analyses to identify rapidly evolving centromere proteins. Our progress represents crucial steps towards understanding the centromere drive conflict but leaves key gaps in our understanding of drive and suppression and centromere protein evolution, which are addressed in this proposal. First, we will determine how selfish centromeres interact with an asymmetric spindle to bias their segregation. Our previous findings suggest a hypothesis that we will test by manipulating microtubule destabilizing activities at centromeres in live cells, using chemical optogenetic approaches that we developed. Second, we will test whether genetically different centromeres differentially recruit centromere proteins, a central but untested component of the centromere drive theory. Using hybrid mouse zygotes with divergent maternal and paternal centromere satellite DNA sequences as a model system, we will determine if rapidly evolving centromere protein interact differentially with different centromere DNA sequences. Third, we will test for reproductive fitness costs associated with functional differences between centromeres, taking advantage of our hybrid mouse model systems in which paired homologous chromosomes in meiosis have divergent centromeres. Fourth, we will test the concept that centromere proteins have evolved to suppress costs due to functional differences between centromeres, which has been the most challenging part of the drive theory to address experimentally. With tractable experimental systems, a mechanistic model for drive and suppression, and molecular evolution analyses of centromere proteins in place, we will address this challenge by testing whether recurrent changes in rapidly evolving centromere proteins have functional implications consistent with our model. Overall, by investigating centromeres in the context of gen...