Investigating the regulation and mechanism of tension-sensors Stu2 & Ndc80c

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Project Summary Proper chromosome segregation is vital for maintenance of eukaryotic genomes, yet the molecular mechanisms underlying this fundamental process remain unclear. This process must occur with absolute fidelity as detrimental aneuploidies result when it goes awry. Aneuploidy is a unifying hallmark of many different cancer types, where it appears to be a therapeutic vulnerability. Understanding the molecular mechanisms of chromosome segregation would give insight into how aneuploidy occurs and how it might be prevented. Our research goal is to understand the regulation and mechanisms of chromosome segregation by probing the function of protein factors responsible for segregating chromosomes. For proper chromosome segregation to occur, duplicated chromosomes must become attached to microtubules originating from opposite cell poles. When a chromosome becomes attached to microtubules from opposing poles in a correct manner, a high force is generated. By contrast, a chromosome attached incorrectly to microtubules from the same cell pole experiences low force. Proteins in the kinetochore complex, which forms the attachment between chromosomes and microtubules, sense and respond to these forces. Kinetochores stabilize “correct” high-force attachments and destabilize “incorrect” low-force attachments through unknown mechanisms, and this activity is vital for proper chromosome segregation. Past work had shown that two conserved eukaryotic factors, Stu2 and its kinetochore receptor the Ndc80 complex (Ndc80c), are required for tension-sensing activity, and we set out to understand the tension-sensing mechanisms of these factors. Using structural and biochemical means, we characterized the physical interaction between Stu2 and Ndc80c and showed that these proteins must interact for proper chromosomes segregation in yeast. These findings are the subject of a recent publication. In this proposal, we will determine how Stu2 and Ndc80c are regulated to control tension-sensing and we will investigate the tension-sensing mechanism by these factors. We will analyze the effects of phosphorylation on Stu2-Ndc80c binding and activity in yeast. Preliminary data showed Stu2 is phosphorylated near the Ndc80c binding site, and that this phosphorylation may affect Ndc80c binding and tension-sensing. Further topology analysis of the Stu2- Ndc80c assembly pointed to an additional interaction interface between these factors that is required for cell viability and possibly tension-sensing. We will also investigate the tension-sensing mechanism of Stu2-Ndc80c by reconstituting the kinetochore microtubule interface and probing it with biophysical methods. These combined approaches will reveal important mechanistic details that are difficult, if not impossible, to obtain by other experimental means. Our past work and preliminary data prime us to be successful in these investigations. Both Stu2 and Ndc80c are mutated in human cancers, with several mutations found at their binding interface, and understanding these mechanisms may provide novel treatment strategies. Overall, this work will increase our understanding of chromosome segregation and how aneuploidy results when this process goes wrong.