The ability of spindle microtubules of to interact dynamically with centromeric chromatin is a critical feature of chromosome segregation and ensures the faithful distribution of genetic material. Errors in this process lead to abnormal chromosome numbers and are a hallmark of cancer and birth defects. The kinetochore is the key cell division organelle that enables high fidelity transmission of genetic information by coupling chromosomes to the plus-ends of spindle microtubules during mitosis and meiosis. Despite its cytological description more than a century ago, little information is available on kinetochore function at a molecular level. Here, I propose to dissect the molecular mechanisms of kinetochore function using the budding yeast Saccharomyces cerevisiae as a model system. My previous work has demonstrated that fundamental aspects of kinetochore organization are conserved throughout evolution. I will use a combination of biochemistry, electron microscopy, in-vitro assays with static and dynamic microtubule substrates as well as yeast cell biology to address fundamental questions of kinetochore function. Specifically, my experiments aim to elucidate 1) the mechanism of phospho-regulation at the kinetochore-microtubule interface 2) the roles of plus-end tracking proteins in chromosome segregation 3) the roles of kinetochore subcomplexes in connecting microtubules and centromeres. Successful completion of the project will help to move the kinetochore field towards a detailed understanding of the molecular mechanisms of chromosome segregation and can open up new perspectives for analyzing the functions of this complex macromolecular machine.